ES2988726T3 - New fluorescent dyes and their uses as biomarkers - Google Patents

Abstract

This application relates to novel fluorescent dyes and their uses as fluorescent labels. The compounds can be used as fluorescent labels for nucleotides in nucleic acid sequencing applications. (Automatic translation with Google Translate, no legal value)

Description

DESCRIPTION

New fluorescent dyes and their uses as biomarkers

Field of invention

The present application relates to novel benzopyran derivatives for use as fluorescent dyes. The compounds can be used as fluorescent labels, particularly for labelling nucleotides in nucleic acid sequencing applications.

Background

Nonradioactive detection of nucleic acids using fluorescent labels is an important technology in molecular biology. Many procedures employed in recombinant DNA technology were previously largely based on the use of nucleotides or polynucleotides radioactively labeled with, for example, 32P. Radioactive compounds allow for the sensitive detection of nucleic acids and other molecules of interest. However, there are serious limitations to the use of radioactive isotopes, such as their expense, limited shelf life, and most importantly, safety concerns. Eliminating the need for radioactive labels improves safety, while reducing the environmental impact and costs associated with, for example, reagent disposal. Methods amenable to nonradioactive fluorescent detection include, but are not limited to, automated DNA sequencing, hybridization methods, real-time detection of polymerase chain reaction products, and immunoassays.

For many applications it is desirable to employ multiple spectrally distinguishable fluorescent labels to achieve independent detection of a plurality of spatially overlapping analytes. In such multiplexed methods, the number of vessels can be reduced to simplify experimental protocols and facilitate the production of application-specific reagent kits. In multicolor automated DNA sequencing, for example, multiplexed fluorescent detection allows the analysis of multiple nucleotide bases in a single electrophoresis lane, thereby increasing throughput compared to single-color methods and reducing uncertainties associated with lane-to-lane electrophoretic mobility variations.

However, multiplexed fluorescent detection can be problematic, and there are a number of important factors that restrict the selection of fluorescent labels. First, it is difficult to find dye compounds whose absorption and emission spectra are spectrally resolved. Furthermore, when multiple fluorescent dyes are used together, simultaneous excitation can be difficult because the absorption bands of the dyes in the different spectral regions are widely separated. Many excitation methods use high-power lasers, and therefore the dye must have sufficient photostability to withstand such laser excitation. A final consideration of particular importance in molecular biology methods is that the fluorescent dyes must be compatible with the reagent chemistries used, such as, for example, solvents and DNA synthesis reagents, buffers, polymerase enzymes, and ligase enzymes.

As sequencing technology advances, a need has developed for additional fluorescent dye compounds, their nucleic acid conjugates and dye sets that satisfy all of the above constraints and are particularly suitable for high-throughput molecular methods such as solid-phase sequencing and the like.

PCT Publication No. WO 2007/135368 describes a class of rhodamine compounds suitable for use as fluorescent labels. The compounds described herein are suitable for use in solid-phase nucleic acid sequencing protocols. Advances in solid-phase nucleic acid sequencing technology and performance have led to further developments and improvements in the molecular design of fluorescent labels, particularly in the context of the interaction between fluorescent reagents and particular nucleic acid sequences.

PCT Publication No. WO 2014/135223 describes a class of rhodamine compounds suitable for use as fluorescent labels.

Fluorescent dye molecules with improved fluorescence properties (such as Stokes shift, fluorescence intensity, position of the fluorescence maximum, and shape of the fluorescence band) can improve the speed and accuracy of nucleic acid sequencing. Stokes shift is an essential aspect in the identification of fluorescent signals in biological applications. For example, the detection of emitted light can be difficult to distinguish from excitation light when fluorophores with absorption and fluorescence maxima very close to each other (i.e., small Stokes shift) are used because the excitation and emission bands largely overlap. In contrast, fluorophores with large Stokes shifts are easy to distinguish, due to the greater separation between the excitation and emission wavelengths. The Stokes shift is especially critical in multiplexed fluorescence applications, because the emission wavelength of one fluorophore may overlap and therefore excite another fluorophore in the same sample. Furthermore, the intensity of the fluorescence signal is especially important when measurements are made in biological buffers containing water and/or at a higher temperature, since the fluorescence of most dyes is significantly lower under such conditions. Furthermore, the nature of the base to which a dye is attached also affects the fluorescence maxima, fluorescence intensity, and other spectral properties of the dye. Sequence-specific interactions between the fluorescent dye and the nucleobase can be tailored by specific design of the fluorescent dyes. Optimization of the structure of fluorescent dyes can improve their fluorescent properties and also improve the efficiency of nucleotide incorporation, reduce the level of sequencing errors and decrease the use of reagents in nucleic acid sequencing and, therefore, its costs.

Described herein are novel benzopyran derivatives and their use as labels for biomolecules, particularly as labels for nucleotides used in nucleic acid sequencing. Improvements can be seen in the higher Stokes shifts of such dyes when prepared as biomolecule conjugates and in the length, intensity and quality of the sequencing read that can be obtained using the new fluorescent compounds.

Summary

The invention provides a nucleotide or oligonucleotide labeled with a fluorescent compound as defined in claims 1 to 9; a kit comprising said labeled nucleotide or oligonucleotide and according to claims 10 to 12; and a sequencing method according to claim 13 and 14. Some embodiments described herein relate to novel benzopyran derivatives of formula (I) or mesomeric forms thereof:

wherein each R1, R2, and R1' is independently selected from the group consisting of H, alkyl, substituted alkyl, alkoxy, alkenyl, alkynyl, haloalkyl, haloalkoxy, alkoxyalkyl, amino, aminoalkyl, aminosulfonyl, halo, cyano, hydroxy, hydroxyalkyl, heteroalkyl, C-carboxy, O-carboxy, C-amido, N-amido, nitro, sulfonyl, sulfo, sulfino, sulfonato, sulfonyl halide, S-sulfonamido, N-sulfonamido, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocyclyl;

alternatively, R1 and R1' together and with the atoms to which they are attached form a ring or ring system selected from the group consisting of optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocyclyl;

each R3 and R4 is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, alkynyl, aminoalkyl, haloalkyl, heteroalkyl, alkoxyalkyl, sulfonyl hydroxide, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted carbocyclyl, and optionally substituted heterocyclyl;

Alternatively, R1 and R3 together with the atoms to which they are attached form a ring or ring system selected from the group consisting of optionally substituted 5-10 membered heteroaryl or optionally substituted 5-10 membered heterocyclyl;

Alternatively, R2 and R4 together with the atoms to which they are attached form a ring or ring system selected from the group consisting of optionally substituted 5-10 membered heteroaryl and optionally substituted 5-10 membered heterocyclyl;

R5 and R6 are independently selected from the group consisting of alkyl, substituted alkyl, alkoxy, alkenyl, alkynyl, haloalkyl, haloalkoxy, alkoxyalkyl, amino, aminoalkyl, aminosulfonyl, halo, cyano, hydroxy, hydroxyalkyl, heteroalkyl, C-carboxy, O-carboxy, C-amido, N-amido, nitro, sulfonyl, sulfo, sulfino, sulfonato, sulfonyl halide, S-sulfonamido, N-sulfonamido, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocyclyl;

R is selected from -OR7 or -NR8R9;

R7 is selected from the group consisting of H, alkyl, substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted carbocyclyl, and optionally substituted heterocyclyl;

each R8 and R9 is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, alkynyl, aminoalkyl, carboxyalkyl, sulfonatoalkyl, haloalkyl, heteroalkyl, alkoxyalkyl, sulfo, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted carbocyclyl, and optionally substituted heterocyclyl;

X is selected from the group consisting of O, S, NR10 and Se;

R10 is selected from the group consisting of H, alkyl, substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted carbocyclyl, and optionally substituted heterocyclyl;

m is an integer selected from 0 to 4; and

n is an integer selected from 0 to 4; provided that

where each R1, R1', and R2 is H; each R3 and R4 is ethyl; each m and n is 0; R is - NHCH(CH3)CH2OH; then X is selected from O, S, or Se;

when each R1, R1', and R2 is H; each R3 and R4 is ethyl; each m and n is 0; R is -OH; then X is selected from S or Se;

when each R1, R1', and R2 is H; each R3 and R4 is ethyl; m is 1 and R5 is methyl; n is 0; R is -OH or -OEt; then X is selected from S, NR10, or Se; and

when each R1, R1', and R2 is H; each R3 and R4 is ethyl; m is 1 and R5 is -S(Oz)Et; n is 0; R is -OH or -OEt; then X is selected from S, NR10, or Se.

Some embodiments described herein relate to a fluorescent compound of formula (V) with a Stokes shift ranging from about 60 nm to about 100 nm:

wherein each R1, R1' and R2 is independently selected from the group consisting of H, alkyl, substituted alkyl, alkoxy, alkenyl, alkynyl, haloalkyl, haloalkoxy, alkoxyalkyl, amino, aminoalkyl, aminosulfonyl, halo, cyano, hydroxy, hydroxyalkyl, heteroalkyl, C-carboxy, O-carboxy, C-amido, N-amido, nitro, sulfonyl, sulfo, sulfino, sulfonato, sulfonyl halide, S-sulfonamido, N-sulfonamido, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heteroaryl and optionally substituted heterocyclyl;

alternatively, R1 and R1' together and with the atoms to which they are attached form a ring or ring system selected from the group consisting of optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocyclyl;

each R3 and R4 is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, alkynyl, aminoalkyl, haloalkyl, heteroalkyl, alkoxyalkyl, sulfo, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted carbocyclyl, and optionally substituted heterocyclyl; alternatively, R1 and R3 together with the atoms to which they are attached form a ring or ring system selected from the group consisting of optionally substituted 5-10 membered heteroaryl or optionally substituted 5-10 membered heterocyclyl;

Alternatively, R2 and R4 together with the atoms to which they are attached form a ring or ring system selected from the group consisting of optionally substituted 5-10 membered heteroaryl and optionally substituted 5-10 membered heterocyclyl;

RHet is a heteroaryl optionally substituted with one or more R5;

each R5 and R6 is independently selected from the group consisting of alkyl, substituted alkyl, alkoxy, alkenyl, alkynyl, haloalkyl, haloalkoxy, alkoxyalkyl, amino, aminoalkyl, aminosulfonyl, halo, cyano, hydroxy, hydroxyalkyl, heteroalkyl, C-carboxy, O-carboxy, C-amido, N-amido, nitro, sulfonyl, sulfo, sulfino, sulfonato, sulfonyl halide, S-sulfonamido, N-sulfonamido, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocyclyl;

R is selected from -OR7 or -NR8R9;

And is selected from O or NH;

R7 is selected from the group consisting of H, alkyl, substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted carbocyclyl, and optionally substituted heterocyclyl;

each R8 and R9 is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, alkynyl, aminoalkyl, carboxyalkyl, sulfonatoalkyl, haloalkyl, heteroalkyl, alkoxyalkyl, sulfo, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted carbocyclyl, and optionally substituted heterocyclyl; and

n is an integer from 0 to 4.

Some descriptions described herein refer to a nucleotide or oligonucleotide labeled with a compound of formula (I) or (V).

Some descriptions described herein relate to kits containing one or more nucleotides where at least one nucleotide is a nucleotide labeled with a fluorescent dye described herein. Some additional descriptions described herein relate to sequencing methods that include incorporating a labeled nucleotide described herein into a sequencing assay and detecting the labeled nucleotide.

Some additional descriptions described herein relate to a method of preparing a compound of formula (Ia), the methods include reacting a compound of formula (IIa)

or formula (IIb)

with a compound of formula (III)

to form

where the variables R1, R1', R2, R3, R4, R5, R6, X, m and n are defined above in the description of compounds of formula (I), and R" is selected from the group consisting of H, optionally substituted alkyl, alkenyl, alkynyl, aminoalkyl, haloalkyl, heteroalkyl, alkoxyalkyl, sulfo, optionally substituted aryl; optionally substituted heteroaryl, optionally substituted carbocyclyl, and optionally substituted heterocyclyl.

Some additional descriptions described herein relate to a method of preparing a compound of formula (Ia), the methods include reacting a compound of formula (IIa)

or formula (IIb)

with a compound of formula (IIIa)

to form

where variables R1, R1', R2, R3, R4, R5, R6, R7, R", X, m and n are defined above.

Some additional disclosures described herein relate to a method of preparing a compound of formula (Ia'), the method including converting a compound of formula (Ia) to a compound of formula (Ia') via carboxylic acid activation:

where the variables R1, R1', R2, R3, R4, R5, R6, R7, R8, R9, X, m and n are defined above in the description of compounds of formula (I).

Some additional disclosures described herein relate to a method of preparing a compound of formula (Ib), the method including converting a compound of formula (Ia) to a compound of formula (Ia') via carboxylic acid activation:

and reacting the compound of formula (Ia') with a primary or secondary amine of formula (IV),

where the variables R1, R1', R2, R3, R4, R5, R6, R7, R8, R9, X, m and n are defined above in the description of compounds of formula (I).

Some additional descriptions described herein relate to a method of preparing a compound of formula (Ib), the methods include reacting a compound of formula (IIa)

or formula (IIb)

with a compound of formula (IIIb)

to form

where variables R1, R1', R2, R3, R4, R5, R6, R7, R", X, m and n are defined above.

Brief description of the drawings

Figure 1 is a line graph illustrating the fluorescent spectra of the benzopyran fluorescent dyes described herein in comparison to commercial dyes with absorption in the same spectral region.

Figures 2A and 2B are graphs illustrating the utility of C-nucleotide labeled with the novel fluorescent dyes as described herein (as shown in black) for sequencing analysis. Figure 3 is a bar graph illustrating the fluorescent intensity of C-nucleotide labeled with the novel fluorescent dyes as described herein when these dyes were excited with blue (460 nm) or green (530 nm) light.

Figure 4 is a bar graph illustrating the fluorescent intensity of C-nucleotide labeled with a novel fluorescent dye as described herein compared to a commercial dye at two different temperatures.

Detailed description

The disclosures described herein relate to novel benzopyran derivatives of the structure of formula (I) or (V) for use as fluorescent dyes. These novel fluorescent dyes have higher Stokes shifts and can be used as fluorescent labels, particularly for nucleotide labeling in nucleic acid sequencing applications. Methods for preparing these fluorescent dyes and subsequent sequencing applications utilizing these dyes are also exemplified. Some embodiments described herein relate to novel benzopyran derivatives of formula (I) or mesomeric forms thereof:

wherein each R1, R2, and R1' is independently selected from the group consisting of H, alkyl, substituted alkyl, alkoxy, alkenyl, alkynyl, haloalkyl, haloalkoxy, alkoxyalkyl, amino, aminoalkyl, aminosulfonyl, halo, cyano, hydroxy, hydroxyalkyl, heteroalkyl, C-carboxy, O-carboxy, C-amido, N-amido, nitro, sulfonyl, sulfo, sulfino, sulfonato, sulfonyl halide, S-sulfonamido, N-sulfonamido, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocyclyl;

alternatively, R1 and R1' together and with the atoms to which they are attached form a ring or ring system selected from the group consisting of optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocyclyl;

each R3 and R4 is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, alkynyl, aminoalkyl, haloalkyl, heteroalkyl, alkoxyalkyl, sulfo, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted carbocyclyl, and optionally substituted heterocyclyl; alternatively, R1 and R3 together with the atoms to which they are attached form a ring or ring system selected from the group consisting of optionally substituted 5-10 membered heteroaryl or optionally substituted 5-10 membered heterocyclyl;

Alternatively, R2 and R4 together with the atoms to which they are attached form a ring or ring system selected from the group consisting of optionally substituted 5-10 membered heteroaryl and optionally substituted 5-10 membered heterocyclyl;

R5 and R6 are independently selected from the group consisting of alkyl, substituted alkyl, alkoxy, alkenyl, alkynyl, haloalkyl, haloalkoxy, alkoxyalkyl, amino, aminoalkyl, aminosulfonyl, halo, cyano, hydroxy, hydroxyalkyl, heteroalkyl, C-carboxy, O-carboxy, C-amido, N-amido, nitro, sulfonyl, sulfo, sulfino, sulfonato, sulfonyl halide, S-sulfonamido, N-sulfonamido, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocyclyl;

R is selected from -OR7 or -NR8R9;

R7 is selected from the group consisting of H, alkyl, substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted carbocyclyl, and optionally substituted heterocyclyl;

each R8 and R9 is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, alkynyl, aminoalkyl, carboxyalkyl, sulfonatoalkyl, haloalkyl, heteroalkyl, alkoxyalkyl, sulfo, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted carbocyclyl, and optionally substituted heterocyclyl;

X is selected from the group consisting of O, S, NR10 and Se;

R10 is selected from the group consisting of H, alkyl, substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted carbocyclyl, and optionally substituted heterocyclyl;

m is an integer selected from 0 to 4; and

n is an integer selected from 0 to 4; provided that

when each R1, R1', and R2 is H; each R3 and R4 is ethyl; each m and n is 0; R is - NHCH(CH3)CH2OH; then X is selected from O, S, or Se;

when each R1, R1', and R2 is H; each R3 and R4 is ethyl; each m and n is 0; R is -OH; then X is selected from S or Se;

when each R1, R1', and R2 is H; each R3 and R4 is ethyl; m is 1 and R5 is methyl; n is 0; R is -OH or -OEt; then X is selected from S, NR10, or Se; and

when each R1, R1', and R2 is H; each R3 and R4 is ethyl; m is 1 and R5 is -S(O2)Et; n is 0; R is -OH or -OEt; then X is selected from S, NR10, or Se.

In some descriptions of compounds of formula (I), when each R1, R1' and R2 is H; each R3 and R4 is ethyl; m is 1; R5 is Cl; then X is selected from S, O or Se, preferably O.

In some embodiments of the compounds of formula (I), the optionally substituted aryl described herein is optionally substituted C6-10 aryl, for example, phenyl. In some embodiments, the optionally substituted heteroaryl described herein is optionally substituted 5-10 membered heteroaryl; more preferably, optionally substituted 5-6 membered heteroaryl. In some embodiments, the optionally substituted carbocyclyl described herein is optionally substituted 3-7 membered carbocyclyl, in particular, 3-7 membered cycloalkyl. In some embodiments, the optionally substituted heterocyclyl described herein is optionally substituted 3-7 membered heterocyclyl, more preferably 5-6 membered heterocyclyl.

In some descriptions of the compounds of formula (I), R is -OR7 and the compound of formula (I) is also represented by formula (Ia'):

In one description, R7 is H and the compound of formula (I) is also represented by formula (Ia):

In some descriptions of compounds of formula (I), R is -NR8R9 and the compound of formula (I) is also represented by formula (Ib):

In one disclosure, each of R<8>and R<9>is H. In some other disclosures, R<8>is H and R<9>is substituted alkyl. In some further embodiments, both R<8>and R<9>are substituted alkyl. In some of these disclosures, the substituted alkyl is selected from alkyl substituted with carboxyl (-C(=O)OH), sulfo (-SO<3>H), or sulfonate (-SO<3">). In some other disclosures, the substituted alkyl is selected from alkyl substituted with a C-amido group.

In some descriptions of compounds of formula (I), (Ia), (Ia'), or (Ib), each of R<1>, R<1'>, and R<2> is H. In some other descriptions, at least one of R<1>, R<1'>, and R<2> is an alkyl.

In some embodiments of the compounds of formula (I), (Ia), (Ia'), or (Ib), each R<3>and R<4>is an alkyl. In some such disclosures, R<3>and/or R<4>may be selected from methyl or ethyl. In some other disclosures, R<3>is H and R<4>is an alkyl. In some disclosures, R<3>and R<4>are methyl.

In some other descriptions of compounds of formula (I), (Ia), (Ia'), or (Ib), R<1> and R<3> together with the atoms to which they are attached form an optionally substituted 3- to 7-membered heterocyclyl, for example, an optionally substituted 6-membered heterocyclyl. In some descriptions, the heterocyclyl ring has one heteroatom (i.e., nitrogen). In some other descriptions, the heterocyclyl ring may have two or more heteroatoms. In one description, the optionally substituted 6-membered heterocyclyl has the structure

where the ----- represents a single bond or a double bond, such that each of the carbon atoms in the ring is neutral and uncharged. In some such disclosures, R4 is selected from H or alkyl. In one disclosure, R4 is ethyl. In some such embodiments, at least one of R1 and R2 is H. In one embodiment, both R1 and R2 are H.

In some other descriptions of compounds of formula (I), (Ia), (Ia'), or (Ib), R2 and R4 together with the atoms to which they are attached form an optionally substituted 3- to 7-membered heterocyclyl, for example, an optionally substituted 6-membered heterocyclyl. In some descriptions, the heterocyclyl ring has one heteroatom (i.e., nitrogen). In some other descriptions, the heterocyclyl ring may have two or more heteroatoms. In one description, the optionally substituted 6-membered heterocyclyl has the structure

where the ----- represents a single bond or a double bond, such that each of the carbon atoms in the ring is neutral and uncharged. In some such descriptions, R3 is selected from H or alkyl. In one description, R3 is ethyl. In some such descriptions, at least one of R1 and R1 is H. In one description, both R1 and R1 are H.

In some alternative descriptions of compounds of formula (I), (Ia), (Ia'), or (Ib), R1 and R3 together with the atoms to which they are attached form an optionally substituted 3- to 7-membered heterocyclyl, for example, an optionally substituted 6-membered heterocyclyl; and R2 and R4 together with the atoms to which they are attached form an optionally substituted 3-7-membered heterocyclyl, for example, an optionally substituted 6-membered heterocyclyl. In some descriptions, the resulting fused heterocyclyl ring system has one heteroatom (i.e., nitrogen). In some other descriptions, the resulting fused heterocyclyl ring system may have two or more heteroatoms. In one description, the resulting fused heterocyclyl ring system has the structure

where the — represents a single bond or a double bond, so that each of the carbon atoms in the ring is neutral and has no charge. In one description, R1 is H.

In some descriptions of compounds of formula (I), (Ia), (Ia') or (Ib), the 3-7 membered heterocyclyl ring formed by R1/R3 and/or R2/R4 are unsubstituted. In some other descriptions, such 3-7 membered heterocyclyl is substituted with one or more alkyls, for example, methyl.

In some descriptions of compounds of formula (I), (Ia), (Ia'), or (Ib), m is 0. In some other descriptions, m is 1. In some such descriptions, R 5 is selected from sulfo, sulfonyl halide, for example, sulfonyl chloride, or aminosulfonyl. In some descriptions, R 5 is halogen. In some descriptions, R 5 is chloro.

In some descriptions of formula (I), (Ia), (Ia'), or (Ib), n is 0.

In any one of the descriptions of the compounds of formula (I), (Ia), (Ia') or (Ib) described herein, X may be S (sulfur). In any one of the descriptions of the compounds of formula (I), (Ia), (Ia') or (Ib) described herein, X may be O (oxygen). In some such descriptions, the compounds of formula (Ia), (Ia') and (Ib) may also be represented by Formulas (Ic), (Ic') and (Id), respectively:

In some specific descriptions, exemplary compounds of formula (I) include:

or mesomeric forms thereof.

In some descriptions of compounds of formula (I), the compound is covalently linked to a nucleotide or oligonucleotide through C(=O)R, where R is -OR7, and where R7 is a substituted alkyl.

In some alternative disclosures, the compound is covalently linked to a nucleotide or oligonucleotide via C(=O)R, wherein R is -NR8R9, and wherein at least one of R8 or R9 comprises at least one functional group that can be used for coupling to biomolecules, for example, one of R8 or R9 is a substituted alkyl comprising at least one carboxyl group.

In some descriptions, the compound of formula (I) is present in one or more mesomeric forms (I-A), (I-B) or (I-C):

Some descriptions described herein refer to a fluorescent compound of formula (V) with a Stokes shift ranging from about 60 nm to about 100 nm:

wherein each R1, R1' and R2 is independently selected from the group consisting of H, alkyl, substituted alkyl, alkoxy, alkenyl, alkynyl, haloalkyl, haloalkoxy, alkoxyalkyl, amino, aminoalkyl, aminosulfonyl, halo, cyano, hydroxy, hydroxyalkyl, heteroalkyl, C-carboxy, O-carboxy, C-amido, N-amido, nitro, sulfonyl, sulfo, sulfino, sulfonato, sulfonyl halide, S-sulfonamido, N-sulfonamido, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heteroaryl and optionally substituted heterocyclyl;

alternatively, R1 and R1' together and with the atoms to which they are attached form a ring or ring system selected from the group consisting of optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocyclyl;

each R3 and R4 is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, alkynyl, aminoalkyl, haloalkyl, heteroalkyl, alkoxyalkyl, sulfo, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted carbocyclyl, and optionally substituted heterocyclyl; alternatively, R1 and R3 together with the atoms to which they are attached form a ring or ring system selected from the group consisting of optionally substituted 5-10 membered heteroaryl or optionally substituted 5-10 membered heterocyclyl;

Alternatively, R2 and R4 together with the atoms to which they are attached form a ring or ring system selected from the group consisting of optionally substituted 5-10 membered heteroaryl and optionally substituted 5-10 membered heterocyclyl;

RHet is a heteroaryl optionally substituted with one or more R5;

each R5 and R6 is independently selected from the group consisting of alkyl, substituted alkyl, alkoxy, alkenyl, alkynyl, haloalkyl, haloalkoxy, alkoxyalkyl, amino, aminoalkyl, aminosulfonyl, halo, cyano, hydroxy, hydroxyalkyl, heteroalkyl, C-carboxy, O-carboxy, C-amido, N-amido, nitro, sulfonyl, sulfo, sulfino, sulfonato, sulfonyl halide, S-sulfonamido, N-sulfonamido, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocyclyl;

R is selected from -OR7 or -NR8R9;

And is selected from O or NH;

R7 is selected from the group consisting of H, alkyl, substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted carbocyclyl, and optionally substituted heterocyclyl;

each R8 and R9 is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, alkynyl, aminoalkyl, carboxyalkyl, sulfonatoalkyl, haloalkyl, heteroalkyl, alkoxyalkyl, sulfo, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted carbocyclyl, and optionally substituted heterocyclyl; and

n is an integer from 0 to 4.

In some descriptions of formula (V), RHet is selected from optionally substituted 5-10 membered heteroaryl. In some of these descriptions, RHet is selected from optionally substituted 9 membered heteroaryl, e.g., optionally substituted benzothiazole. In one description, RHet is optionally substituted 2-benzothiazolyl:

In one description, RHet is optionally substituted 2-benzoxazolyl with the structure

In some such descriptions, RHet is substituted with one or more substituents selected from sulfo, sulfonyl halide or aminosulfonyl. In some such descriptions, RHet is substituted with one or more halogens. In some such descriptions, RHet is substituted with a chlorine.

In some descriptions, R is -OR7 and compounds of formula (V) are also represented by formula (Va):

In one description, R7 is H. In another description, R7 is substituted alkyl.

In some descriptions, R is -NR8R9 and compounds of formula (V) are also represented by formula (Vb):

In one disclosure, each of R<8> and R<9> is H. In some other disclosures, R<8> is H and R<9> is substituted alkyl. In some further embodiments, both R<8> and R<9> are substituted alkyl. In some such disclosures, the substituted alkyl is selected from alkyl substituted with carboxyl (-C(=O)OH) or sulfo (-SO<3>H) or sulfonate (-SO<3">). In some other disclosures, the substituted alkyl is selected from alkyl substituted with a C-amido group.

In some descriptions of compounds of formula (V), (Va) or (Vb), Y is O.

In some descriptions of compounds of formula (V), (Va), or (Vb), each of R<1>, R<1'>, and R<2> is H. In some other descriptions, at least one of R<1>, R<1'>, and R<2> is an alkyl.

In some descriptions of compounds of formula (V), (Va) or (Vb), each R<3>and R<4>is an alkyl. In some such descriptions, R<3>and/or R<4>may be selected from methyl or ethyl. In some other descriptions, R<3>is H and R<4>is an alkyl.

In some other descriptions of compounds of formula (V), (Va) or (Vb), R<1> and R<3> together with the atoms to which they are attached form an optionally substituted 3-7 membered heterocyclyl, for example, an optionally substituted 6 membered heterocyclyl. In some descriptions, the heterocyclyl ring has one heteroatom (i.e. nitrogen). In some other descriptions, the heterocyclyl ring may have two or more heteroatoms. In one description, the optionally substituted 6 membered heterocyclyl has the structure

where e l-----represents a single bond or a double bond, such that each of the carbon atoms in the ring is neutral and uncharged. In some such disclosures, R4 is selected from H or alkyl. In one disclosure, R4 is ethyl. In some further embodiments, at least one of R1 and R2 is H. In one disclosure, both R1 and R2 are H.

In some other descriptions of compounds of formula (V), (Va) or (Vb), R2 and R4 together with the atoms to which they are attached form an optionally substituted 3- to 7-membered heterocyclyl, for example, an optionally substituted 6-membered heterocyclyl. In some descriptions, the heterocyclyl ring has one heteroatom (i.e., nitrogen). In some other descriptions, the heterocyclyl ring may have two or more heteroatoms. In one description, the optionally substituted 6-membered heterocyclyl has the structure

where e l-----represents a single bond or a double bond, such that each of the carbon atoms in the ring is neutral and uncharged. In some such descriptions, R3 is selected from H or alkyl. In one description, R3 is ethyl. In some additional descriptions, at least one of R1 and R1 is H. In one description, both R1 and R1 are H.

In some alternative descriptions of compounds of formula (V), (Va) or (Vb), R1 and R3 together with the atoms to which they are attached form an optionally substituted 3-7 membered heterocyclyl, for example, an optionally substituted 6 membered heterocyclyl; and R2 and R4 together with the atoms to which they are attached form an optionally substituted 3-7 membered heterocyclyl, for example, an optionally substituted 6 membered heterocyclyl. In some descriptions, the resulting fused heterocyclyl ring system has one heteroatom (i.e., nitrogen). In some other descriptions, the resulting fused heterocyclyl ring system may have two or more heteroatoms. In one description, the resulting fused heterocyclyl ring system has the structure

where the ----- represents a single bond or a double bond, such that each of the carbon atoms in the ring is neutral and has no charge. In one description, R1 is H.

In some descriptions of compounds of formula (V), (Va) or (Vb), the 3-7 membered heterocyclyl ring formed by R1/R3 and/or R2/R4 are unsubstituted. In some other descriptions, such 3-7 membered heterocyclyl is substituted with one or more alkyls, for example, methyl.

In some descriptions of compounds of formula (V), (Va) or (Vb), n is 0.

In some specific descriptions of formula (V), (Va) or (Vb), exemplary compounds of formula (V) include compounds I-1 through I-15 as described herein.

In some disclosures, the fluorescent compound of formula (V) is covalently linked to a nucleotide or oligonucleotide via C(=O)R, where R is -OR7, and where R7 is a substituted alkyl.

In some alternative descriptions, the fluorescent compound of formula (V) is covalently linked to a nucleotide or oligonucleotide through C(=O)R, where R is -NR8R9, and wherein at least one of R8 or R9 is a substituted alkyl.

Definitions

The section headings used herein are for organizational purposes only and should not be construed as a limitation of the subject matter described.

Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one skilled in the art. Furthermore, the use of the term “including” as well as other forms, such as “include”, “includes” and “included”, is not limiting. Furthermore, the use of the term “having”, as well as other forms, such as “have”, “has” and “had”, is not limiting. As used herein, whether in a transitional expression or in the body of the claim, the terms “consisting of” and “consisting of” are to be construed as having an open-ended meaning. That is, the terms are to be construed as synonyms of the terms “having at least” or “including at least”. When used in the context of a process, the term “consisting of” means that the process includes at least the recited steps, but may include additional steps. When used in the context of a compound, composition, or device, the term “consisting of” means that the compound, composition, or device includes at least the recited features or components, but may also include additional features or components.

As used herein, common organic abbreviations are defined as follows: Ac Acetyl

Ac2O Acetic anhydride

ac. Aqueous

BOC or Boc tert-butyloxycarbonyl

BOP (Benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate

cat. Catalyst

°C temperature in degrees Celsius

dATP deoxyadenosine triphosphate

dCTP deoxycytidine triphosphate

dGTP deoxyguanosine triphosphate

dTTP deoxythymidine triphosphate

ddNTP(s) dideoxynucleotide(s)

DCM methylene chloride

DMA dimethylacetamide

DMF dimethylformamide

Et ethyl

EtOAc ethyl acetate

ffN Fully functional nucleotide conjugate

ffC Fully Functional Cytidine Conjugate

g gram(s)

h or hr hour or hours

IPA isopropyl alcohol

LCMS liquid chromatography-mass spectrometry

MeCN acetonitrile

mL milliliter(s)

PG protective group

Phenyl Ph

ppt precipitate

PyBOP (benzotriazol-1-yloxy) tripyrrolidinomial hexafluorophosphate

at room temperature

SBS sequencing by synthesis

TEA triethylamine

TEAB tetraethylammonium bicarbonate

TFA trifluoroacetic acid

Tert, tertiary t

THF tetrahydrofuran

TLC thin layer chromatography

TSTU O-(N-succinimidyl) N,N,N',N'-tetramethyluronium tetrafluoroborate

|jL microliter(s)

As used herein, the term “covalently bonded” or “covalently linked” refers to a chemical bond conformation that is characterized by the sharing of electron pairs between atoms. For example, a “covalently bonded polymer sheet”, when used in reference to the surface of a substrate, refers to a polymer sheet that forms chemical bonds with the functionalized surface of a substrate, as opposed to bonding to the surface through other means, for example, adhesion or electrostatic interaction. It will be appreciated that polymers that are covalently bonded to a surface may also be bonded through means other than covalent bonding.

The term “halogen” or “halo”, as used herein, means any one of the radiostable atoms in column 7 of the Periodic Table of the Elements, e.g., fluorine, chlorine, bromine or iodine, with fluorine and chlorine being preferred.

As used herein, “alkyl” refers to a straight or branched hydrocarbon chain that is fully saturated (i.e., containing no double or triple bonds). The alkyl group may have from 1 to 20 carbon atoms (wherever appearing herein, a numerical range such as “1 to 20” refers to each integer in the given range; e.g., “1 to 20 carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms, although the present definition also encompasses the occurrence of the term “alkyl” where no numerical range is designated). The alkyl group may also be a mid-size alkyl having from 1 to 9 carbon atoms. The alkyl group could be a lower alkyl having from 1 to 6 carbon atoms. The alkyl group may be designated as “C<1-4> alkyl” or similar designations. By way of example only, “C<1-6> alkyl” indicates that there are one to four carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl and t-butyl. Typical alkyl groups include, but are not in any way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl and the like.

As used herein, “alkoxy” refers to the formula -OR where R is an alkyl as defined above, such as “C<1-9>alkoxy”, which includes, but is not limited to, methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, iso-butoxy, sec-butoxy, and tert-butoxy, and the like.

As used herein, “alkenyl” refers to a straight or branched hydrocarbon chain containing one or more double bonds. The alkenyl group may have from 2 to 20 carbon atoms, although the present definition also encompasses the occurrence of the term “alkenyl” where no numerical range is designated. The alkenyl group may also be a mid-sized alkenyl having from 2 to 9 carbon atoms. The alkenyl group could be a lower alkenyl having from 2 to 6 carbon atoms. The alkenyl group may be designated as “C<2-6> alkenyl” or similar designations. By way of example only, “C<2-6> alkenyl” indicates that there are two to six carbon atoms in the alkenyl chain, i.e., the alkenyl chain is selected from the group consisting of ethenyl, propen-1-yl, propen-2-yl, propen-3-yl, buten-1-yl, buten-2-yl, buten-3-yl, buten-4-yl, 1-methylpropen-1-yl, 2-methylpropen-1-yl, 1-ethylethen-1-yl, 2-methylpropen-3-yl, buta-1,3-dienyl, buta-1,2-dienyl, and buta-1,2-dien-4-yl. Typical alkenyl groups include, but are not in any way limited to, ethenyl, propenyl, butenyl, pentenyl, and hexenyl, and the like.

As used herein, “alkynyl” refers to a straight or branched hydrocarbon chain containing one or more triple bonds. The alkynyl group may have from 2 to 20 carbon atoms, although the present definition also encompasses the occurrence of the term “alkynyl” where no numerical range is designated. The alkynyl group may also be a mid-sized alkynyl having from 2 to 9 carbon atoms. The alkynyl group could be a lower alkynyl having from 2 to 6 carbon atoms. The alkynyl group may be designated as “C<2-6> alkynyl” or similar designations. By way of example only, “C<2-6> alkynyl” indicates that there are two to six carbon atoms in the alkynyl chain, i.e., the alkynyl chain is selected from the group consisting of ethynyl, propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-3-yl, butyn-4-yl and 2-butynyl. Typical alkynyl groups include, but are by no means limited to, ethynyl, propynyl, butynyl, pentynyl and hexynyl, and the like.

As used herein, “heteroalkyl” refers to a straight or branched hydrocarbon chain containing one or more heteroatoms, i.e., an element other than carbon, including, but not limited to, nitrogen, oxygen, and sulfur, in the basic molecular structure of the chain. The heteroalkyl group may have from 1 to 20 carbon atoms, although this definition also encompasses the occurrence of the term “heteroalkyl” when no numerical range is designated. The heteroalkyl group may also be a mid-size heteroalkyl having from 1 to 9 carbon atoms. The heteroalkyl group could be a lower heteroalkyl having from 1 to 6 carbon atoms. The heteroalkyl group may be designated as “C<1-6> heteroalkyl” or similar designations. The heteroalkyl group may contain one or more heteroatoms. Just as an example, “C<1-6> heteroalkyl” indicates that there are one to six carbon atoms in the heteroalkyl chain and additionally one or more heteroatoms in the basic molecular structure of the chain.

The term “aromatic” refers to a ring or ring system having a conjugated pi electron system and includes carbocyclic (e.g., phenyl) and heterocyclic (e.g., pyridine) aromatic groups. The term includes monocyclic or fused-ring polycyclic groups (i.e., rings that share adjacent pairs of atoms), provided that the entire ring system is aromatic.

As used herein, “aryl” refers to an aromatic ring or ring system (i.e., two or more fused rings that share two adjacent carbon atoms) that contain only carbon in the basic molecular structure of the ring. When aryl is a ring system, each ring in the system is aromatic. The aryl group may have from 6 to 18 carbon atoms, although the present definition also encompasses the occurrence of the term “aryl” when no numerical range is designated. In some embodiments, the aryl group has from 6 to 10 carbon atoms. The aryl group may be designated as “C<6-10>aryl”, “C<6>aryl”, or “C<10>aryl” or similar designations. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, fluorenyl, azulenyl, anthryl, phenanthryl, pyrenyl, biphenyl, and terphenyl.

An “aralkyl” or “arylalkyl” is an aryl group connected, as a substituent, through an alkylene group, such as “C<7-14> aralkyl” and the like, including, but not limited to, benzyl, 2-phenylethyl, 3-phenylpropyl, and naphthylalkyl. In some instances, the alkylene group is a lower alkylene group (i.e., a C<1-6> alkylene group).

As used herein, “heteroaryl” refers to an aromatic ring or ring system (i.e., two or more fused rings that share two adjacent atoms) that contains one or more heteroatoms, i.e., an element other than carbon, including but not limited to nitrogen, oxygen, and sulfur, in the basic molecular structure of the ring. When heteroaryl is a ring system, each ring in the system is aromatic. The heteroaryl group may have 5-18 ring members (i.e., the number of atoms that form the basic molecular structure of the ring, which includes carbon atoms and heteroatoms), although the present definition also encompasses the occurrence of the term “heteroaryl” when no numerical range is designated. In some embodiments, the heteroaryl group has 5 to 10 ring members or 5 to 7 ring members. The heteroaryl group may be designated as “5-7 membered heteroaryl”, “5-10 membered heteroaryl”, or similar designations. Examples of heteroaryl rings include, but are not limited to, furyl, thienyl, phthalazinyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, quinolinyl, isoquinyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, indolyl, isoindolyl, and benzothienyl.

A “heteroaralkyl” or “heteroarylalkyl” is a heteroaryl group connected, as a substituent, through an alkylene group. Examples include, but are not limited to, 2-thienylmethyl, 3-thienylmethyl, furylmethyl, thienylethyl, pyrrolylalkyl, pyridylalkyl, isoxazolylalkyl, and imidazolylalkyl. In some instances, the alkylene group is a lower alkylene group (i.e., a C<1-6> alkylene group).

As used herein, “carbocyclyl” refers to a non-aromatic cyclic ring or ring system(s) that contains only carbon atoms in the basic molecular structure of the ring system. When carbocyclyl is a ring system, two or more rings may be joined in a fused, bridged, or spirally connected manner. Carbocyclyls may have any degree of saturation, provided that at least one ring in a ring system is non-aromatic. Thus, carbocyclyls include cycloalkyls, cycloalkenyls, and cycloalkynyls. The carbocyclyl group may have from 3 to 20 carbon atoms, although the present definition also encompasses the occurrence of the term “carbocyclyl” when no numerical range is designated. The carbocyclyl group may also be a medium-sized carbocyclyl having from 3 to 10 carbon atoms. The carbocyclyl group may be a carbocyclyl having 3 to 6 carbon atoms. The carbocyclyl group may be designated as “C<3-6>carbocyclyl” or similar designations. Examples of carbocyclyl rings include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, 2,3-dihydro-indene, bicyclo[2.2.2]octanyl, adamantyl, and spiro[4.4]nonanyl.

As used herein, “cycloalkyl” means a fully saturated carbocyclyl ring or ring system. Examples include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

As used herein, “heterocyclyl” refers to a non-aromatic cyclic ring or ring system that contains at least one heteroatom in the basic molecular structure of the ring. Heterocyclyls may be joined together in a fused, bridged, or spiro-connected manner. Heterocyclyls may have any degree of saturation as long as at least one ring in the ring system is non-aromatic. The heteroatom(s) may be present in an aromatic or non-aromatic ring within the ring system. The heterocyclyl group may have 3 to 20 ring members (i.e., the number of atoms that form the basic molecular structure of the ring, which includes carbon atoms and heteroatoms), although the present definition also encompasses the occurrence of the term “heterocyclyl” where no numerical range is designated. The heterocyclyl group may also be a medium-sized heterocyclyl having 3 to 10 ring members. The heterocyclyl group may also be a heterocyclyl having 3 to 6 ring members. The heterocyclyl group may be designated as “3-6 membered heterocyclyl” or similar designations. In preferred six-membered monocyclic heterocyclyls, the heteroatom(s) is(are) selected from one to three of O, N, or S, and among preferred five-membered monocyclic heterocyclyls, the heteroatom(s) is(are) selected from one or two heteroatoms selected from O, N, or S. Examples of heterocyclyl rings include, but are not limited to, azepinyl, acridinyl, carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl, imidazolidinyl, morpholinyl, oxiranyl, oxeanyl, thiepanyl, piperidinyl, piperazinyl, dioxoperazinyl, pyrrolidinyl, pyrrolidonyl, pyrrolidionyl, 4-piperidonyl, pyrazolinyl, pyrazolidinyl, 1,3-dioxinyl, 1,3-dioxanyl, 1,4-dioxinyl, 1,4-dioxanyl, 1,3-oxatianyl, 1,4-oxatiinyl, 1,4-oxatianyl, 2H-1,2-oxazinyl, trioxanyl, hexahydro-1,3,5-triazinyl, 1,3-dioxolyl, 1,3-dioxolanyl, 1,3-dithiolyl, 1,3-dithiolanyl, isoxazolinyl, isoxazolidinyl, oxazolinyl, oxazolidinyl, oxazolidinonyl, thiazolinyl, thiazolidinyl, 1,3-oxathiolanyl, indolinyl, isoindolinyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydro-1,4-thiazinyl, thiamorpholinyl, dihydrobenzofuranyl, benzimidazolidinyl, and tetrahydroquinoline.

An “O-carboxy” group refers to a “-OC(=O)R” group in which R is selected from hydrogen, C<1-6> alkyl, C<2-6> alkenyl, C<2-6> alkynyl, C<3-7> carbocyclyl, C<6-10> aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein.

A “C-carboxy” group refers to a “-C(=O)OR” group wherein R is selected from hydrogen, C<1-6> alkyl, C<2-6> alkenyl, C<2-6> alkynyl, C<3-7> carbocyclyl, C<6-10> aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein. A non-limiting example includes carboxyl (i.e., -C(=O)OH).

A “cyano” group refers to a “-CN” group.

A “sulfonyl” group refers to a “-SO<2>R” group in which R is selected from hydrogen, C<1-6> alkyl, C<2-6> alkenyl, C<2-6> alkynyl, C<3-7> carbocyclyl, C<6-10> aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein.

A “sulfo” or “sulfonyl hydroxide” group refers to a “-S(=O)<2>-OH” group.

A “sulfino” group refers to a “-S(=O)OH” group.

A “sulfonate” group refers to -SO<3 '>.

A “sulfonyl halide” group refers to a “-S(=O)<2>-X” group, where X is a halide.

An “S-sulfonamido” group refers to a “-SO<2>NR<a>R<b>” group wherein R<a>and R<b>are each independently selected from hydrogen, C<1-6>alkyl, C<2-6>alkenyl, C<2-6>alkynyl, C<3-7>carbocyclyl, C<6-10>aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein.

An “N-sulfonamido” group refers to a “-N(R<a>)SO<2>R<b>” group wherein R<a>and R<b>are each independently selected from hydrogen, C<1-6>alkyl, C<2-6>alkenyl, C<2-6>alkynyl, C<3-7>carbocyclyl, C<6-10>aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein.

A “C-amido” group refers to a “-C(=O)NR<a>R<b>” group wherein R<a>and R<b>are each independently selected from hydrogen, C<1-6>alkyl, C<2-6>alkenyl, C<2-6>alkynyl, C<3-7>carbocyclyl, C<6-10>aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein.

An “N-amido” group refers to a “-N(R<a>)C(=O)R<b>” group wherein R<a>and R<b>are each independently selected from hydrogen, C<1-6>alkyl, C<2-6>alkenyl, C<2-6>alkynyl, C<3-7>carbocyclyl, C<6-10>aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein.

An “amino” group refers to a “-NR<a>R<b>” group where R<a>and R<b>are each independently selected from hydrogen, C<1-6>alkyl, C<2-6>alkenyl, C<2-6>alkynyl, C<3-7>carbocyclyl, C<6-10>aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein. A non-limiting example includes free amino (i.e., -NH<2>).

An “aminoalkyl” group refers to an amino group connected through an alkylene group.

An “aminosulfonyl” group refers to a “-S(=O)<2>NH<2>” group.

An “alkoxyalkyl” group refers to an alkoxy group connected through an alkylene group, such as “C<2-8>alkoxyalkyl” and the like.

As used herein, a substituted group is derived from the original unsubstituted group in which there has been an exchange of one or more hydrogen atoms for another atom or group. Unless otherwise indicated, when a group is considered to be “substituted” it means that the group is substituted with one or more substituents independently selected from C<1>-C<6>alkyl, C<1>-C<6>alkenyl, C<1>-C<6>alkynyl, C<1>-C<6>heteroalkyl, C<3>-C<7>carbocyclyl (optionally substituted with halo, C<1>-C<6>alkyl, C<1>-C<6>alkoxy, C<1>-C<6>haloalkyl, and C<1>-C<6>haloalkoxy), C<3>-C<7>carbocyclyl-C<1>-C<6>alkyl(optionally substituted with halo, C<1>-C<6>alkyl, C<1>-C<6>alkoxy, haloalkyl, and C<1>-C<6>haloalkoxy). C<1>-C<6>and haloalkoxy C<1>-C<6>), 3-10 membered heterocyclyl (optionally substituted with halo, C<1>-C<6>alkyl, C<1>-C<6>alkoxy, C<1>-C<6>haloalkyl and C<1>-C<6>haloalkoxy), 3-10 membered heterocyclyl-C<1>-C<6>alkyl (optionally substituted with halo, C<1>-C<6>alkyl, C<1>-C<6>alkoxy, C<1>-C<6>haloalkyl and C<1>-C<6>haloalkoxy), aryl (optionally substituted with halo, C<1>-C<6>alkyl, C<1>-C<6>alkoxy, C<1>-C<6>haloalkyl and halo(C<1>-C<6>alkoxy), aryl(C<1>-C<6>alkyl) (optionally substituted with halo, C<1>-C<6>alkyl, C<1>-C<6>alkoxy, C<1>-C<6>haloalkyl, and C<1>-C<6>haloalkoxy), 5-10 membered heteroaryl (optionally substituted with halo, C<1>-C<6>alkyl, C<1>-C<6>alkoxy, C<1>-C<6>haloalkyl, and C<1>-C<6>haloalkoxy), 5-10 membered heteroaryl(C<1>-C<6>alkyl) (optionally substituted with halo, C<1>-C<6>alkyl, C<1>-C<6>alkoxy, C<1>-C<6>haloalkyl, and C<1>-C<6>haloalkoxy), 5-10 membered heteroaryl(C<1>-C<6>alkyl) (optionally substituted with halo, C<1>-C<6>alkyl, C<1>-C<6>alkoxy, C<1>-C<6>haloalkyl, and halo(C<1>-C<6>)alkoxy, halo, cyano, hydroxy, (C<1>-C<6>)alkoxy, (C<1>-C<6>)alkoxy-alkyl (i.e. ether), aryloxy, sulfhydryl (mercapto), halo(C<1>-C<6>)alkyl (e.g. e.g., -CF<3>), halo-alkoxy (C<1>-C<6>) (e.g., -OCF<3>), C<1>-C<6>alkylthio, arylthio, amino, amino-alkyl (C<1>-C<6>), nitro, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, acyl, cyanato, isocyanato, thiocyanato, isothiocyanato, sulfinyl, sulfo, sulfino, sulfonato and oxo (=O). Whenever a group is described as “optionally substituted”, that group may be substituted with the above substituents.

As one skilled in the art will understand, if a compound contains positively or negatively charged substituent groups, for example, SO<3 ->, it may also contain a negatively or positively charged counterion, such that the compound as a whole is neutral.

It should be understood that certain radical naming conventions may include either a monoradical or a diradical, depending on the context. For example, when a substituent requires two points of attachment to the remainder of the molecule, the substituent is understood to be a diradical. For example, a substituent identified as alkyl that requires two points of attachment includes diradicals such as -CH<2>-, -CH<2>CH<2>-, -CH<2>CH(CH<3>)CH<2>-, and the like. Other radical naming conventions clearly indicate that the radical is a diradical, such as “alkylene” or “alkenylene”.

When two “adjacent” R groups are said to form a ring “together with the atom to which they are attached”, it means that the collective unit of the atoms, intervening bonds, and the two R groups are the ring in question. For example, when the following substructure is present:

and R1 and R2 are defined as selected from the group consisting of hydrogen and alkyl, or R1 and R2, together with the atoms to which they are attached, form an aryl or carbocyclyl, it is understood that R1 and R2 may be selected from hydrogen or alkyl or, alternatively, the substructure has the structure:

Where A is an aryl or carbocyclyl ring containing the double bond depicted.

Labeled nucleotides

The dye compounds described herein are suitable for binding to substrate moieties. Substrate moieties can be virtually any molecule or substance to which the fluorescent dyes described herein can be conjugated, and by way of non-limiting example, can include nucleosides, nucleotides, polynucleotides, carbohydrates, ligands, particles, solid surfaces, organic and inorganic polymers, and combinations or assemblies thereof, such as chromosomes, nuclei, living cells, and the like. The dyes can be conjugated to an optional linker by a variety of means including hydrophobic attraction, ionic attraction, and covalent attachment. Particularly, the dyes are conjugated to the substrate by covalent attachment. More particularly, covalent attachment is via a linker group. In some instances, such labeled nucleotides are also referred to as “modified nucleotides.”

A particularly useful application of the novel long Stokes shift fluorescent dyes, as described herein, is for labeling biomolecules, e.g., nucleotides or oligonucleotides. Some embodiments of the present application are directed to a nucleotide or oligonucleotide labeled with the novel fluorescent compounds, as described herein.

Coupling to biomolecules may be via the -C(=O)R moiety of the compound of formula (I) or (V). In some disclosures, R is -OR7 and R7 is a substituted alkyl, which may be used to couple to the amino group of biomolecules. In one disclosure, the -C(O)R moiety may be an activated ester residue more suitable for increased amide/peptide bond formation. The term “activated ester”, as used herein, refers to a derivative of the carboxy group that is capable of reacting under mild conditions with, for example, a compound containing an amino group. Non-limiting examples of activated esters include, but are not limited to, p-nitrophenyl, pentafluorophenyl, and succinimido esters. In some other descriptions, R is -NR8R9 and at least one of R8 or R9 contains at least one functional group that can be used for coupling to biomolecules, for example, one of R8 or R9 is a substituted alkyl comprising at least one carboxyl.

In some embodiments, the dye compounds may be covalently linked to oligonucleotides or nucleotides via the nucleotide base. For example, the labeled nucleotide or oligonucleotide may have the label attached to the C5 position of a pyrimidine base or the C7 position of a 7-deazapurine base via a linker moiety. The labeled nucleotide or oligonucleotide may also have a 3'-OH blocking group covalently linked to the ribose or deoxyribose sugar of the nucleotide.

Linkers

The dye compounds, as described herein, may include a reactive linker group at one of the substituent positions of the covalent attachment of the compound to another molecule. Reactive linker groups are moieties capable of forming a covalent bond. In a particular embodiment, the linker may be a cleavable linker. The use of the term “cleavable linker” is not intended to imply that the entire linker is required to be removed. The cleavage site may be located at a position on the linker that ensures that part of the linker remains attached to the dye and/or the remainder of the substrate after cleavage. Cleavable linkers may be, by way of non-limiting example, electrophilic cleavable linkers, nucleophilic cleavable linkers, photocleavable linkers, cleavable under reducing conditions (e.g. disulfide- or azide-containing linkers), oxidative conditions, cleavable through the use of guard-capture linkers, and may be cleaved by elimination mechanisms. The use of a cleavable linker to attach the dye compound to a substrate moiety ensures that the label can, if necessary, be removed after detection, thus avoiding any interfering signal at later stages.

Non-limiting examples of linker groups include those described in PCT Publication No. WO2004/018493, which connect nucleotide bases to labels such as, for example, the novel fluorescent compounds described herein. These linkers can be cleaved using water-soluble phosphines or water-soluble transition metal catalysts formed from a transition metal and at least partially water-soluble ligands. In aqueous solution, the latter at least partially forms water-soluble transition metal complexes. Additional suitable linkers that can be used include those described in PCT Publication Nos. WO2004/018493 and WO 2007/020457. It was discovered that by altering, and in particular increasing, the length of the linker between a fluorescent dye (fluorophore) and the guanine base, by introducing a polyethylene glycol spacer group, it is possible to increase the intensity of the fluorescence, compared to the same fluorophore attached to the guanine base through other linkages known in the art. The design of the linkers, and especially their increased length, also allows for improved brightness of fluorophores attached to the guanine bases of guanosine nucleotides when incorporated into polynucleotides such as DNA. Therefore, when the dye is used in any analysis method requiring the detection of a fluorescent dye label coupled to a guanine-containing nucleotide, it is advantageous if the linker comprises a spacer group of the formula -((CH2)2O)n-, where n is an integer between 2 and 50, as described in WO 2007/020457.

Nucleosides and nucleotides may be labeled on sugar or nucleobase sites. As will be understood by one skilled in the art, a “nucleotide” consists of a nitrogenous base, a sugar, and one or more phosphate groups. In RNA, the sugar is a ribose, and in DNA it is a deoxyribose, i.e., a sugar lacking a hydroxyl group that is present in ribose. The nitrogenous base is a derivative of purine or pyrimidine. Purines are adenine (A) and guanine (G), and pyrimidines are cytosine (C) and thymine (T) or in the context of RNA, uracil (U). The C-1 atom of deoxyribose binds to the N-1 of a pyrimidine or the N-9 of a purine. A nucleotide is also a phosphate ester of a nucleoside, having the esterification at the hydroxyl group attached to C-3 or C-5 of the sugar. Nucleotides are generally mono-, di- or triphosphates.

A “nucleoside” is structurally similar to a nucleotide, but is missing the phosphate moieties. An example of a nucleoside analogue would be one in which the tag is attached to the base and there is no phosphate group attached to the sugar molecule.

Although the base is generally referred to as a “purine” or “pyrimidine,” the skilled artisan will appreciate that available derivatives and analogues do not alter the ability of the nucleotide or nucleoside to undergo Watson-Crick base pairing. “Derivative” or “analogue” is meant to refer to a compound or molecule whose core structure is the same as or closely resembles that of a parent compound, but which has a chemical or physical modification, such as a different or additional side group, that allows the derived nucleotide or nucleoside to be attached to another molecule. For example, the base may be a deazopurine. Derivatives must be capable of undergoing Watson-Crick base pairing. “Derivative” and “analogue” also refer to a synthetic nucleotide or derivative of a nucleoside that has modified base moieties and/or modified sugar moieties. Such derivatives and analogues are discussed, for example, in Scheit, Nucleotide analogs (John Wiley & Son, 1980) and Uhlman et al., Chemical Reviews 90:543-584, 1990. Nucleotide analogues may also consist of modified phosphodiester linkages including phosphorothioate, phosphorodithioate, alkylphosphonate, phosphoranilide, phosphoramidate, and the like linkages.

The dye may be attached to any position on the nucleotide base, via a linker, as long as Watson-Crick base pairing can still take place. Certain nucleobase labeling sites include the C5 position of a pyrimidine base or the C7 position of a 7-deazapurine base. As described above, a linker group may be used to covalently attach a dye to the nucleoside or nucleotide.

In particular, the labeled nucleoside or nucleotide may be enzymatically incorporated and enzymatically extendable. Accordingly, a linker moiety may be of sufficient length to connect the nucleotide to the compound such that the compound does not significantly interfere with the overall binding and recognition of the nucleotide by a nucleic acid replication enzyme. The linker may therefore also consist of a spacer unit. Spacer units, for example, the base of the nucleotides of a region or a cleavage tag, distance the nucleotides from the nucleotides to the compound.

Nucleosides or nucleotides labeled with the novel fluorescent dyes described herein may have the formula:

where Dye is a dye compound, B is a nucleobase, such as uracil, thymine, cytosine, adenine, guanine, and the like, and L is an optional linking group which may or may not be present. R' can be H, monophosphate, diphosphate, triphosphate, thiophosphate, a phosphate ester analog, -O- linked to a reactive phosphorus-containing group, or -O- protected by a blocking group. R" can be H, OH, a phosphoramidite, or a 3'-OH blocking group, and Rm is H or OH; where R" is phosphoramidite, R' is an acid-cleavable hydroxyl protecting group allowing subsequent monomer coupling under automated synthesis conditions.

In some cases, the blocking group is separate and independent of the dye compound, i.e., not attached to it. Alternatively, the dye may consist of all or part of the 3'-OH blocking group. Thus, R" may be a 3'-OH blocking group which may or may not consist of the dye compound. In further alternative embodiments, there is no blocking group on the 3' carbon of the pentose sugar and the dye (or the dye and linker) attached to the base, for example, may be of a size or structure sufficient to act as a block for the incorporation of an additional nucleotide from a point other than the 3' site. Thus, the blockage may be due to steric hindrance or may be due to a combination of size, charge and structure.

The use of a blocking group allows the polymerization to be controlled, such as stopping extension when a modified nucleotide is incorporated. If the blocking effect is reversible, for example, by way of non-limiting example, changing chemical conditions or by removing a chemical block, extension can be stopped at certain points and then allowed to continue. Non-limiting examples of 3'-OH blocking groups include those described in WO 2004/018497 and WO2014/139596. For example, the blocking group can be azidomethyl (-CH<2>N<3>) or substituted azidomethyl (e.g., -CH(CHF<2>)N<3>or CH(CH<2>F)N<3>) or allyl.

In a particular embodiment, the linker and the blocking group are present and are separate moieties that are both cleavable under substantially similar conditions. Therefore, the deprotection and deblocking processes may be more efficient, since only a single treatment will be required to remove both the dye compound and the blocking group.

The present embodiment also directs the expression of polynucleotides incorporating dye compounds described herein. Such polynucleotides may be DNA or RNA consisting of, respectively, deoxyribonucleotides or ribonucleotides linked in a phosphodiester bond. The polynucleotides may consist of naturally occurring nucleotides, non-natural (or modified) nucleotides other than the labeled nucleotides described herein, or any combination thereof, provided that at least one nucleotide labeled with a dye compound is present, according to the present application. The polynucleotides may also include non-natural backbone segments and/or non-nucleotide chemical modifications. Chimeric structures composed of mixtures of ribonucleotides and deoxyribonucleotides consisting of at least one labeled nucleotide are also contemplated.

Non-limiting exemplary labeled nucleotides, as described herein, include:

and

Kits

Some embodiments described herein are kits that include nucleosides and/or nucleotides labeled with the novel fluorescent dyes described herein. Such kits will generally include at least one nucleotide or nucleoside labeled with a dye along with at least one additional component. The additional components may be additional modified or unmodified nucleotides or nucleosides. For example, dye-labeled nucleotides may be supplied in combination with unlabeled or native nucleotides, and/or with fluorescently labeled nucleotides or any combination thereof. Combinations of nucleotides may be provided as separate individual components or as mixtures of nucleotides. In some embodiments, the kits comprise one or more nucleotides wherein at least one nucleotide is a nucleotide labeled with a novel fluorescent compound described herein. The kits may comprise two or more labeled nucleotides. The nucleotides may be labeled with two or more fluorescent labels. Two or more of the tags can be excited using a single excitation source, which may be a laser.

The kits may contain four labeled nucleotides, of which the first of the four nucleotides is labeled with one compound, as described herein, and the second, third, and fourth nucleotides are each labeled with a different compound, wherein each compound has a distinct fluorescence maximum and each of the compounds is distinguishable from the other three compounds. The kits may be such that two or more of the compounds have similar absorbance maxima but different Stokes shifts.

The fluorescent dye compounds, nucleotides or labeled kits described herein may be used in sequencing, expression analysis, hybridization analysis, genetic analysis, RNA analysis or protein binding assays. Use may be in an automated sequencing instrument. The sequencing instrument may contain two lasers operating at different wavelengths.

When the kits comprise a plurality, particularly two, more particularly four, nucleotides labeled with a dye compound, the different nucleotides may be labeled with different dye compounds, or one may be dark, with no dye compounds. When the different nucleotides are labeled with different dye compounds, it is a feature of the kits that said dye compounds are spectrally distinguishable fluorescent dyes. As used herein, the term "spectrally distinguishable fluorescent dyes" refers to fluorescent dyes that emit fluorescent energy at wavelengths that can be distinguished by fluorescent detection equipment (e.g., a commercial capillary-based DNA sequencing platform) when two or more of said dyes are present in a sample. When two nucleotides labeled with fluorescent dye compounds are supplied in kit form, the spectrally distinguishable fluorescent dyes may be excited at the same wavelength, such as, for example, by the same laser in some embodiments. When four nucleotides labeled with fluorescent dye compounds are supplied in kit form, two of the spectrally distinguishable fluorescent dyes may be excited at one wavelength and the other two spectrally distinguishable dyes may be excited at another wavelength in some embodiments. Particular excitation wavelengths are about 460 nm.

In a single embodiment, a kit comprises a nucleotide labeled with a compound described herein and a second nucleotide labeled with a second dye wherein the dyes have a difference in absorbance maxima of at least 10 nm, particularly from 20 nm to 50 nm. More particularly, the two dye compounds have Stokes shifts of between 15-40 nm or between 20-40 nm. As used herein, the term “Stokes shift” is the difference between the positions of the band maxima of the absorption and emission spectra of the same electronic transition.

In a further embodiment, said kit further comprises two other labels with fluorescent dyes wherein said dyes are excited by the same lasers from about 460 nm to about 540 nm.

In an alternative embodiment, the kits may contain nucleotides where the same base is labeled with two different compounds. A first nucleotide may be labeled with a compound described herein. A second nucleotide may be labeled with a spectrally distinct compound, for example, a “red” dye that absorbs greater than 600 nm. A third nucleotide may be labeled as a mixture of the fluorescent dye compound described herein and the spectrally distinct compound, and the fourth nucleotide may be “dark” and contain no label. In simple terms, therefore, nucleotides 1-4 may be labeled as “green,” “reddish,” “red/green,” and dark. To simplify the instrumentation, four nucleotides may be labeled with two dyes excited with a single laser, and thus the labeling of nucleotides 1-4 may be “green 1,” “green 2,” “green 1/green 2,” and dark.

In other embodiments, the kits may include a polymerase enzyme capable of catalyzing the incorporation of the nucleotides into a polynucleotide. Other components to be included in such kits may include buffers and the like. Nucleotides labeled with the novel fluorescent dyes described herein, and other nucleotide components including mixtures of different nucleotides, may be provided in the kit in a concentrated form to be diluted prior to use. In such embodiments, a suitable dilution buffer may also be included.

Sequencing methods

The nucleotides (or nucleosides) comprising a novel fluorescent dye described herein may be used in any analysis method that requires the detection of a fluorescent label attached to a nucleotide or nucleoside, either by itself or incorporated into or associated with a larger molecular structure or conjugate. Some embodiments of the present application are directed to sequencing methods that include: (a) incorporating at least one labeled nucleotide, as described herein, into a polynucleotide; and (b) detecting the labeled nucleotide(s) incorporated into the polynucleotide by detecting the fluorescent signal from the novel fluorescent dye attached to said modified nucleotide(s).

In some embodiments, at least one labeled nucleotide is incorporated into a polynucleotide in the synthesis phase by the action of a polymerase enzyme. However, other methods of incorporating labeled nucleotides into polynucleotides, such as chemical oligonucleotide synthesis or ligation of labeled oligonucleotides to unlabeled oligonucleotides, are not excluded. Thus, the term “incorporating” a nucleotide into a polynucleotide encompasses the synthesis of polynucleotides by chemical methods as well as enzymatic methods.

In all embodiments of the methods, the detection step may be carried out while the polynucleotide chain into which the labeled nucleotides are incorporated is hybridized to a template strand, or after a denaturation step in which the two strands are separated. Additional steps, for example, chemical or enzymatic reaction steps or purification steps, may be included between the synthesis step and the detection step. In particular, the target strand incorporating the labeled nucleotide(s) may be isolated or purified and then further processed or used in a subsequent analysis. By way of example, target polynucleotides labeled with modified nucleotides as described herein in a synthetic step may subsequently be used as labeled probes or primers. In other embodiments, the product of the synthesis step (a) may be subjected to additional reaction steps and, if desired, the product of these subsequent steps is purified or isolated.

Suitable conditions for the synthesis step will be well known to those familiar with the standard techniques of molecular biology. In one embodiment, the synthesis step may be analogous to a standard primer extension reaction using nucleotide precursors, including nucleotides modified according to the present embodiment, to form an extended target strand complementary to the template strand in the presence of a suitable polymerase enzyme. In other embodiments, the synthesis step may form part of an amplification reaction that generates a labeled double-stranded amplification product composed of hybridized complementary strands derived from copying the target and template polynucleotide strands. Other exemplary "synthetic" steps include nick translation, strand displacement polymerization, random priming DNA labeling, etc. The polymerase enzyme used in the synthesis step should be capable of catalyzing the incorporation of modified nucleotides according to the present disclosure. Otherwise, the precise nature of the polymerase is not particularly limited, but may depend on the conditions of the synthetic reaction. By way of example, if the synthetic reaction is carried out by thermocycling, a thermostable polymerase is required, whereas this may not be essential for standard primer extension reactions. Suitable thermostable polymerases that are capable of incorporating modified nucleotides according to the present disclosure include those described in WO 2005/024010 or WO 2006/120433. In synthetic reactions carried out at lower temperatures, such as 37 °C, the polymerase enzymes need not necessarily be the thermostable polymerases; therefore, the choice of polymerase will depend on a number of factors, such as reaction temperature, pH, strand displacement activity and the like.

In specific non-limiting embodiments, the modified nucleotides or nucleosides labeled with the novel longer Stokes shift fluorescent dyes according to the present application may be used in a method of nucleic acid sequencing, resequencing, whole genome sequencing, single nucleotide polymorphism scoring, any other application involving the detection of the modified nucleotide or nucleoside when incorporated into a polynucleotide, or any other application requiring the use of polynucleotides labeled with the modified nucleotides consisting of fluorescent dyes according to the present application.

In a particular embodiment, the present application provides the use of modified nucleotides consisting of dye compounds described herein in a polynucleotide “sequencing by synthesis” reaction. Sequencing by synthesis generally involves the sequential addition of one or more nucleotides or oligonucleotides to a growing polynucleotide chain in the 5' to 3' direction using a polymerase or ligase to form an extended polynucleotide chain complementary to the template nucleic acid to be sequenced. The identity of the base present in one or more of the added nucleotides is determined in a detection or “imaging” step. The identity of the added base can be determined after each nucleotide incorporation step. The sequence of the template can be inferred using conventional Watson-Crick base pairing rules. The use of the dye-labeled modified nucleotides according to the present embodiment for determining single base identity may be useful, for example, in scoring single nucleotide polymorphisms, and such single base extension reactions are within the scope of this application.

In one embodiment, the sequence of a template polynucleotide is determined by detecting the incorporation of one or more nucleotides into a nascent chain complementary to the template polynucleotide to be sequenced through the detection of fluorescent labels attached to the incorporated nucleotide(s). The sequencing template polynucleotide is primed with a suitable primer (or prepared as a hairpin construct which will contain the primer as part of the hairpin), and the nascent chain is extended in a stepwise manner by the addition of nucleotides to the 3' end of the primer in a polymerase-catalyzed reaction.

In particular, each of the different nucleotide triphosphates (A, T, G and C) may be labelled with a unique fluorophore and also consist of a blocking group at the 3' position to prevent uncontrolled polymerisation. Alternatively, one of the four nucleotides may be unlabelled (dark). The polymerase enzyme incorporates a nucleotide into the nascent chain complementary to the template polynucleotide, and the blocking group prevents further nucleotide incorporation. Any unincorporated nucleotide is removed and the fluorescent signal from each incorporated nucleotide is "read out" optically by suitable means, such as a charge-coupled device employing laser excitation and suitable emission filters. The 3' blocking group and fluorescent dye compounds are then removed (deprotected), particularly by the same chemical or enzymatic method, to expose the nascent chain to further nucleotide incorporation. Typically, the identity of the incorporated nucleotide will be determined after each incorporation step, but this is not strictly essential. Similarly, US Patent No. 5,302,509 describes a method for sequencing polynucleotides immobilized on a solid support. The method is based on the incorporation of fluorescently labeled 3'-blocked A, G, C, and T nucleotides into a growing strand complementary to the immobilized polynucleotide, in the presence of DNA polymerase. The polymerase incorporates a base complementary to the target polynucleotide, but further additions are prevented by the 3'-blocking group. The tag of the incorporated nucleotide can then be determined and the blocking group removed by chemical cleavage to allow further polymerization to occur. The nucleic acid template to be sequenced in a sequencing-by-synthesis reaction can be any polynucleotide that is desired to be sequenced. The nucleic acid template for a sequencing reaction will typically consist of a double-stranded region having a free 3'-hydroxyl group that serves as a primer or initiation point for the addition of further nucleotides in the sequencing reaction. The region of the template to be sequenced will overhang this free 3' hydroxyl group on the complementary strand. The overhanging region of the template to be sequenced may be single-stranded, but may be double-stranded, provided that a "nick" is present on the strand complementary to the template strand to be sequenced to provide a free 3' OH group at the start of the sequencing reaction. In such embodiments, sequencing may proceed by strand displacement. In certain embodiments, a primer carrying the free 3' hydroxyl group may be added as a separate component (e.g., a short oligonucleotide) that hybridizes to a single-stranded region of the template to be sequenced. Alternatively, the primer and the template strand to be sequenced may be part of a partially self-complementary nucleic acid strand capable of forming an intramolecular duplex, such as a hairpin loop structure. Hairpin polynucleotides and methods by which they may be coupled to solid supports are described in PCT Publication Nos. WO 2001/057248 and WO 2005/047301. Nucleotides are successively added to the free 3'-hydroxyl group, resulting in the synthesis of a polynucleotide chain in the 5' to 3' direction. The nature of the base that has been added can be determined, particularly but not necessarily, after each nucleotide addition, thus providing sequence information on the nucleic acid template. The term "incorporation" of a nucleotide into a nucleic acid chain (or polynucleotide) in this context refers to the attachment of the nucleotide to the free 3' hydroxyl group of the nucleic acid chain through the formation of a phosphodiester bond with the 5' phosphate group of the nucleotide.

The nucleic acid template to be sequenced may be DNA or RNA, or even a hybrid molecule composed of deoxynucleotides and ribonucleotides. The nucleic acid template may consist of naturally occurring and/or non-naturally occurring nucleotides and natural or non-natural backbone linkages, provided that these do not prevent copying of the template in the sequencing reaction.

In certain embodiments, the nucleic acid template to be sequenced may be attached to a solid support via any suitable attachment method known in the art, e.g., by covalent attachment. In certain embodiments, the template polynucleotides may be directly attached to a solid support (e.g., a silica-based support). However, in other embodiments, the surface of the solid support may be modified in some way to allow for direct covalent attachment of the template polynucleotides, or the template polynucleotides may be immobilized via a hydrogel or polyelectrolyte multilayer, which may be non-covalently attached to the solid support.

Arrays in which polynucleotides have been directly attached to silica-based supports are described for example in PCT Publication No. WO 2000/006770, where polynucleotides are immobilized on a glass support by reaction between a pendant epoxide group on the glass with an internal amino group on the polynucleotide. Furthermore, PCT Publication No. WO2005/047301 describes arrays of polynucleotides attached to a solid support, for example, for use in preparing single molecule arrays by the reaction of a sulfur-based nucleophile with the solid support. Yet another example of solid-supported template polynucleotides is where the template polynucleotides are attached to hydrogel supported on silica-based or other solid supports. Silica-based supports are typically used to support hydrogels and hydrogel matrices as described in PCT Publication Nos. WO 00/31148, WO 01/01143, WO02/12566, WO 03/014392, WO 00/53812 and US Patent No. 6,465,178.

A particular surface to which template polynucleotides can be immobilized is a polyacrylamide hydrogel. Polyacrylamide hydrogels are described in the prior art, some of which have been discussed above. Specific hydrogels that can be used in the present application include those described in WO 2005/065814 and US Publication 2014/0079923. In a single embodiment, the hydrogel is PAZAM (poly(N-(5-azidoacetamyl)acrylamide-co-acrylamide).

DNA template molecules may be attached to microspheres or microparticles to effect sequencing, for example as described in US Patent No. 6,172,218. Other examples of the preparation of microsphere libraries where each microsphere contains different DNA sequences can be found in Margulies et al., Nature 437, 376-380 (2005); Shendure et al., Science. 309(5741): 1728-1732 (2005). Sequencing of such microsphere arrays using nucleotides as described is within the scope of the present application.

The template or templates to be sequenced may form part of an "array" on a solid support, in which case the array may take any convenient form. Thus, the method of the present embodiment is applicable to all types of "high density" arrays, including single molecule arrays, arrayed arrays, and microsphere arrays. The modified nucleotides labeled with dye compounds of the present application may be used in sequencing templates on essentially any type of array consisting of the immobilization of nucleic acid molecules on a solid support, and more particularly any type of high density array. However, modified nucleotides labeled with the novel fluorescent dyes described herein are particularly advantageous in the context of sequencing arrayed arrays.

In multiplex or arrayed arrays, distinct regions of the array consist of several polynucleotide template molecules. The term “arrayed” refers to an array in which distinct regions or zones of the array consist of several polynucleotide molecules that cannot be individually resolved by optical means. Depending on how the array is formed, each zone in the array may consist of several copies of a single individual polynucleotide molecule or even several copies of a small number of different polynucleotide molecules (e.g., several copies of two complementary nucleic acid strands). Multiplex or arrayed arrays of polynucleotides of nucleic acid molecules can be produced using techniques generally known in the art. By way of example, WO 98/44151 and WO 00/18957 describe methods of nucleic acid amplification where both the template and amplification products remain immobilized on a solid support to form arrays composed of clusters or “colonies” of immobilized nucleic acid molecules. Nucleic acid molecules present in the pooled arrays prepared according to these methods are suitable templates for sequencing using the modified nucleotides labeled with the novel fluorescent dyes described herein.

The modified nucleotides labeled with dye compounds of the present application are also useful in sequencing templates on single molecule arrays. The term “single molecule array” or “SMA” as used herein refers to a population of polynucleotide molecules, distributed (or arrayed) on a solid support, wherein the separation of any single polynucleotide from all others in the population is such that individual resolution of the polynucleotides is possible. Therefore, target nucleic acid molecules immobilized on the surface of the solid support must be capable of being resolved by optical means. This means that within the resolvable area of the particular imaging device used, there must be one or more distinct signals, each of which represents a polynucleotide.

This may be achieved where the spacing between adjacent polynucleotide molecules in the array is at least 100 nm, more particularly at least 250 nm, still more particularly at least 300 nm, even more particularly at least 350 nm. Each molecule may thus be individually resolved and detected as a single-molecule fluorescent spot, and the fluorescence of said single-molecule fluorescent spot also exhibits single-step photobleaching.

The terms “singly resolved” and “single resolution” are used herein to specify that, when visualized, it is possible to distinguish a molecule in the array from its neighboring molecules. The separation between individual molecules in the array will be determined, in part, by the particular technique used to resolve the individual molecules. The general characteristics of single molecule arrays will be understood with reference to PCT Publication Nos. WO 2000/006770 and WO 2001/057248. Although one application of the modified nucleotides herein is in sequencing-by-synthesis reactions, the utility of such labeled nucleotides is not limited to such methods. Indeed, the nucleotides may be advantageously used in any sequencing methodology that requires the detection of fluorescent labels attached to nucleotides incorporated into a polynucleotide.

In particular, modified nucleotides labeled with the dye compounds of the present application can be used in automated fluorescent sequencing protocols, particularly, fluorescent dye terminator cycle sequencing based on the chain termination sequencing method of Sanger et al. Such methods generally employ enzymes and cycle sequencing to incorporate fluorescently labeled dideoxynucleotides into a primer extension sequencing reaction. So-called “Sanger sequencing methods” and related (Sanger-type) protocols rely on random chain termination with labeled dideoxynucleotides.

Therefore, the present embodiment also encompasses modified nucleotides labeled with dye compounds as described herein which are dideoxynucleotides lacking hydroxyl groups at both the 3' and 2' positions, said modified dideoxynucleotides being suitable for use in Sanger-type sequencing methods and the like.

Preparation methods

Some additional descriptions described herein relate to a method of preparing a compound of formula (Ia), the methods include reacting a compound of formula (IIa)

or formula (IIb)

with a compound of formula (III)

to form

where the variables R1, R1', R2, R3, R4, R5, R6, X, m and n are defined above in the description of compounds of formula (I), and R" is selected from the group consisting of H, optionally substituted alkyl, alkenyl, alkynyl, aminoalkyl, haloalkyl, heteroalkyl, alkoxyalkyl, sulfo, optionally substituted aryl; optionally substituted heteroaryl, optionally substituted carbocyclyl, and optionally substituted heterocyclyl.

Some additional descriptions described herein relate to a method of preparing a compound of formula (Ia), the methods include reacting a compound of formula (IIa)

or formula (IIb)

with a compound of the formula (Illa)

to form

where variables R1, R1', R2, R3, R4, R5, R6, R7, R", X, m and n are defined above.

Some additional disclosures described herein relate to a method of preparing a compound of formula (Ia'), the method including converting a compound of formula (Ia) to a compound of formula (Ia') via carboxylic acid activation:

where the variables R1, R1', R2, R3, R4, R5, R6, R7, R8, R9, X, m and n are defined above in the description of compounds of formula (I).

Some additional disclosures described herein relate to a method of preparing a compound of formula (Ib), the method including converting a compound of formula (la) to a compound of formula (Ia') via carboxylic acid activation:

and reacting the compound of formula (Ia') with a primary or secondary amine of formula (IV),

where the variables R1, R1', R2, R3, R4, R5, R6, R7, R8, R9, X, m and n are defined above in the description of compounds of formula (I).

Some additional descriptions described herein relate to a method of preparing a compound of formula (Ib), the methods include reacting a compound of formula (IIa)

or formula (IIb)

with a compound of formula (IIIb)

to form

where variables R1, R1', R2, R3, R4, R5, R6, R7, R", X, m and n are defined above.

The preparation of compounds of formula (Ia) may be achieved by reacting starting materials of formula (IIa) or (IIb) with the intermediate compound of formula (III) preferably in a one to one molar ratio in an organic solvent with or without a catalyst. Both organic catalysts (e.g. trifluoroacetic acid or methanesulfonic acid) and inorganic catalysts (e.g. phosphoric or sulfuric acid) may be used. In some disclosures, the preparation may be performed without a solvent or by using a catalyst as a solvent.

The preparation of compounds of formula (Ia') or (Ib) may be achieved by first acidic activation of the starting materials of formula (Ia) in an organic solvent at room temperature using, for example, carbodiimide, O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium salts, BOP or PyBOP as an activating reagent followed by reaction with appropriate hydroxylated derivatives HOR7 or amine of formula (IV) for the coupling step.

Compounds of formula (V), (Va) or (Vb) may be prepared following synthetic schemes similar to those described above in the preparation of compounds of formula (Ia), (Ia') or (Ib).

The compounds of formula (I) can be used in some organic reactions, for example in electrophilic aromatic substitution reactions as starting materials for the synthesis of new dyes. For example, it was previously shown by Alan S. Waggoner (Bioconjugate Chemistry, 1993, 4(2): 105-111; see also patent publications US-2015/0274976) that some modifications of cyanine dye molecules by sulfonation reaction can enhance the fluorescent properties of such derivatives.

Sulfonation of compounds (I) can be carried out by reaction with sulfur trioxide with sulfur trioxide derivatives or solutions, for example, in sulfuric acid.

Sulfochlorination of compounds (I) can be carried out by reaction with chlorosulfonic acid.

Compounds of formula (I) containing sulfonic or chlorosulfonic groups can be further modified, for example, by reacting them with ammonia, primary or secondary amines of formula (IV):

where the variables R1, R1', R2, R3, R4, R5, R6, R7, R8, R9, X, n are defined above in the description of compounds of formula (I).

Examples

Other embodiments are described in more detail in the following examples, which are not intended to limit the scope of the claims.

General procedure for the synthesis of compounds of formula (Ia)

A mixture of ethyl benzothiazolyl-2-acetic acid (2.2 g, 0.01 mol) and an appropriate hydroxybenzoylbenzoic acid derivative (0.011 mol) was dissolved in concentrated sulfuric acid (5 mL). This reaction mixture was stirred at room temperature for 1 hour and then heated, for example, at 80-120 °C for 1-2 hours until the reaction was complete. The reaction mixture was poured onto ice (about 50 g) and the product was filtered, washed with water. In many cases, the final products do not need any further purification.

General procedure for the synthesis of compounds of formula (Ia')

A compound of formula (Ia) (0.001 mol) was dissolved in anhydrous DMF (1.5 ml). To this solution was added carbodiimide (0.0012 mol) or another activating reagent. This reaction mixture was stirred at room temperature for 3 hours and then appropriate hydroxy derivatives R7OH were added. The reaction mixture was stirred overnight, then filtered and poured onto ice (ca. 50 g). The product was filtered, washed with water. Yield: 55-75 %. In many cases, the final products do not need any further purification.

General procedure for the synthesis of compounds of formula (Ib)

A compound of formula (Ia) (0.001 mol) was dissolved in a suitable anhydrous organic solvent (DMF, 1.5 mL). To this solution, TSTU, BOP or PyBOP was added as an activating reagent. This reaction mixture was stirred at room temperature for about 20 minutes and then the appropriate amine NHR8R9 was added. The reaction mixture was stirred overnight, filtered and the excess activating reagent was quenched with a 0.1 M solution of TEAB in water. The solvents were evaporated in vacuo and the residue was purified by HPLC. Yield: 65-75 %.

Example 1

2-[3-(Benzothiazol-2-yl)-9-ethyl-2-oxo-6,7,8,9-tetrahydro-2H-pyrano[3,2-g]quinolin-4-yl]benzoic acid (compound 1-3)

Sulfuric acid (2 mL) was placed in a round bottom flask, then cooled to approximately 0-5 °C and 0.325 g (1.0 mmol, 1 eq) of 2-(1-ethyl-7-hydroxy-1,2,3,4-tetrahydroquinoline-6-carbonyl)benzoic acid was added with stirring followed by the addition of 0.3 g (1.4 mmol, 1.4 eq) of ethyl 2-(benzothiazol-2-yl)acetate. This reaction mixture was stirred at room temperature (approximately 20 °C) for 30 minutes, then heated for 1.5 hours while stirring at 100 °C. The progress of the reaction was monitored by TLC (DCM-MeOH, 10%) and by LCMS.

The reaction mixture was left at room temperature (ca. 20 °C) for 0.5 h and then poured into a mixture of crushed ice (ca. 100 g) and sodium acetate (ca. 5 g). After 1 h, the product was filtered and washed with water until neutral, then the filtrate was air dried. Yield: 0.35 g (0.73 mmol, 73 %). The final product was used without further purification. MS (DUIS): MW calculated 482.13. Found: (-) 481 (M-1), (+) 483 (M+1). 1H NMR (400 MHz, DMSO-da) 5 12.71 (s, 1H), 8.11 - 8.02 (m, 1H), 7.96 (dd, J = 7.4, 1.6 Hz, 1H), 7.56 (tt, J = 7.5, 5.8 Hz, 2H), 7.40 (d, J = 7.2 Hz, 1H), 7.39 - 7.25 (m, 2H), 7.16 (dd, J = 6.6, 2.1 Hz, 1H), 6.67 (s, 1H), 6.35 (s, 1H), 3.48 (q, J = 7.0 Hz, 2H), 3.36 (t, J = 5.7 Hz, 2H), 2.65 - 2.55 (m, 2H), 1.79 (q, J = 6.3 Hz, 2H), 1.15 (t, J = 7.0 Hz, 3H).

Example 2

2-[10-(benzothiazol-2-yl)-1,1,7,7-tetramethyl-11-oxo-2,3,6,7-tetrahydro-1 H,5H,11 H-pyran[2, 3-f]pyrido[3,2,1-yl]quinolin-9-yl]benzoic acid (compound I-5)

A mixture of 0.75 g (1.90 mmol, 1 eq) of 2-(8-hydroxy-1,1,7,7-tetramethyl-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-yl]quinoline-9-carbonyl)benzoic acid and 0.44 g (1.99 mmol, 1.04 eq) of ethyl 2-(benzothiazol-2-yl)acetate was added with stirring to concentrated sulfuric acid (7 mL) at room temperature (about 20 °C). This reaction mixture was stirred at room temperature for 30 minutes, then heated for 2.5 hours while stirring at 120 °C. The progress of the reaction was monitored by TLC (DCM-MeOH, 10%) and by LCMS. The reaction mixture was left at room temperature (ca. 20 °C) for 0.5 h and then poured into a mixture of crushed ice (ca. 50 g) and sodium acetate (ca. 5 g). After 1 h, the product was filtered and washed with water to neutrality and air dried. Yield: 0.58 g (1.05 mmol, 55 %). The final product was used without further purification. MS (DUIS): MW calculated 550.19. Found: (-) 549 (M-1 ).

Example 3

2-[3-(Benzothiazol-2-yl)-7-(ethylamino)-6-methyl-2-oxo-2H-chromen-4-yl]benzoic acid (compound I-8)

Sulfuric acid (5 mL) was placed in a round bottom flask, then cooled to approximately 0-5 °C and 0.7 g (2.34 mmol, 1 eq) of 2-(4-(ethylamino)-2-hydroxy-5-methylbenzoyl)benzoic acid was added with stirring followed by the addition of 0.7 g (3.16 mmol, 1.35 eq) of ethyl 2-(benzothiazol-2-yl)acetate. This reaction mixture was stirred for 30 minutes at room temperature (approximately 20 °C) and then heated for 1 hour while stirring at 95 °C. The progress of the reaction was monitored by TLC (DCM-MeOH, 10%) and by LCMS.

The reaction mixture was left at room temperature (ca. 20 °C) for 0.5 h and then poured into a mixture of crushed ice (ca. 100 g) and sodium acetate (ca. 5 g). After 1 h, the product was filtered and washed with water until neutral and air dried. Yield: 1.01 g (2.22 mmol, 95 %). The final product was used without further purification. MS (DUIS): MW calculated 456.11. Found: (-) 455 (M-1), (+) 457 (M+1). 1H NMR (400 MHz, methanol-d4) 58.19 - 8.12 (m, 1H), 7.92 - 7.78 (m, 1H), 7.64 (dd, J = 8.3, 1.1 Hz, 1H), 7.61 - 7.48 (m, 2H), 7.43 - 7.36 (m, 1H), 7.36 - 7.26 (m, 1H), 7.21 - 7.14 (m, 1H), 6.60 (s, 1H), 6.56 (d, J = 1.1 Hz, 1H), 3.41 - 3.34 (m, 2H), 2.03 (s, 3H), 1.38 - 1.27 (m, 3H).

Example 4-1

2-[10-(benzothiazol-2-yl)-11-imino-2,3,6,7-tetrahydro-1H,5H,11H-pyrano[2,3-f]pyrido[3,2,1- ilquinolin-9-ylbenzoic acid (compound I-4A)

Sulfuric acid (10 mL) was placed in a round-bottom flask, then 1 g (2.96 mmol, 1 eq) of 2-(8-hydroxy-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-yl]quinoline-9-carbonyl)benzoic acid was added with stirring followed by the addition of 0.57 g (3.26 mmol, 1.1 eq) of 2-(benzothiazol-2-yl)acetonitrile. This reaction mixture was stirred at room temperature (about 20 °C) for 30 minutes and then heated for 1.5 hours while stirring at 80 °C and 1 hour at 90 °C. The progress of the reaction was monitored by TLC (DCM-MeOH, 10%) and by LCMS.

The reaction mixture was left at room temperature (ca. 20 °C) for 0.5 h and then poured onto crushed ice mixture (ca. 100 g). After 0.5 h, the product was filtered and washed with water until neutral and air dried. Yield: 0.8 g (1.62 mmol, 55 %). The final product was used without further purification. MS (DUIS): MW calculated 493.15. Found: (-) 492 (M-1), (+) 494 (M+1).

Example 4-2

2-[10-(benzothiazol-2-yl)-11 -oxo-2,3,6,7-tetrahydro-1 H,5H,11 H-pyrano[2,3-f]pyrido[3,2, 1-yl]quinolin-9-yl]benzoic acid (compound I-4).

The suspension of imine (I-4A) from the previous step (example 4-1) and water (25 ml) was stirred 5 hours at 80 °C. At the end of this time, the red color of the starting material almost disappeared and a very strong fluorescence developed. The progress of the reaction was monitored by TLC (DCM-MeOH, 10 %) and by LCMS. The reaction mixture was left at room temperature (ca. 20 °C) for 0.5 hour and then the product was filtered and washed with water and air dried. Yield: 0.77 g (1.56 mmol, 96 %). The final product was used without further purification. MS (DUIS): calculated MW 494.13. Found: (-) 493 (M-1), (+) 495 (M+1).

Example 5

2-[10-(benzothiazol-2-yl)-11 -oxo-2,3,6,7-tetrahydro-1 H,5H,11 H-pyrano[2,3-f]pyrido[3,2, 1-ylquinolin-9-ylbenzoic acid (compound I-4)

Sulfuric acid (10 mL) was placed in a round-bottom flask and then 1 g (2.96 mmol, 1 eq) of 2-(8-hydroxy-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-yl]quinoline-9-carbonyl)benzoic acid was added with stirring followed by the addition of 0.57 g (3.26 mmol, 1.1 eq) of 2-cyanomethylbenzothiazole. This reaction mixture was stirred at room temperature (about 20 °C) for 30 minutes, then heated for 2 hours while stirring at 95 °C. The reaction mixture was allowed to cool to room temperature (about 20 °C) and then poured into a mixture of water and crushed ice (about 75 g). This mixture was stirred overnight at room temperature and the product was filtered and washed with water until neutral and air dried. The final product was used without further purification. Yield: 1.09 g (2.21 mmol, 75 %). MS (DUIS): calculated MW 494.13. Found: (-) 493 (M-1), (+) 495 (M+1).

Example 6

2-[3-(Benzothiazol-2-yl)-7-(dimethylamino)-2-oxo-2H-chromen-4-yl]benzoic acid (compound I-2)

A mixture of 0.285 g (1.0 mmol, 1 eq) of 2-[4-(dimethylamino)-2-hydroxybenzoyl]benzoic acid and 0.243 g (1.1 mmol, 1.1 eq) of ethyl 2-(benzothiazol-2-yl)acetate was added with stirring to sulfuric acid (5 mL) at room temperature (ca. 20 °C). This reaction mixture was stirred at room temperature for 3 hours, then heated for 4 hours while stirring at 100 °C. The progress of the reaction was monitored by TLC (DCM-MeOH, 10%) and by LCMS. The reaction mixture was left at room temperature (ca. 20 °C) overnight and then poured into a mixture of water with crushed ice (ca. 100 g) and sodium acetate (ca. 5 g). After 1 hour, the product was filtered and washed with water until neutral and air dried. Yield: 0.429 g (0.96 mmol, 97 %). The final product was used without further purification. MS (DUIS): MW calculated 442.10. Found: (-) 441 (M-1). 1H NMR (400 MHz, DMSO-d6) 5 12.70 (s, 1H), 8.14 - 8.04 (m, 1H), 8.04 - 7.94 (m, 1H), 7.58 (h, J = 6.5 Hz, 2H), 7.43 (d, J = 7.8 Hz, 1H), 7.40 - 7.29 (m, 2H), 7.19 (dd, J = 7.2, 1.7 Hz, 1H), 6.78 - 6.63 (m, 3H), 3.07 (s, 6H).

Example 7-1

2-[3-(Benzothiazol-2-yl)-7-(diethylamino)-2-oxo-2H-chromen-4-yl]benzoic acid (compound 1-1).

Sulfuric acid (8 mL) was placed in a round bottom flask, then 0.25 g (0.8 mmol, 1 eq) of 2-[4-(diethylamino)-2-hydroxybenzoyl]benzoic acid was added with stirring followed by the addition of 0.14 g (0.8 mmol, 1 eq) of 2-cyanomethylbenzothiazole. The reaction mixture was stirred at room temperature (about 20 °C) for 30 minutes and then heated for 2 hours while stirring at 95 °C. This reaction mixture was poured into ice water (about 75 g). At this stage, the corresponding imine intermediate (I-1A) is formed and can be isolated by filtration. In water or in organic solvents with the presence of water and particularly under acidic conditions further hydrolysis of the imine intermediate can be achieved.

This mixture was stirred overnight at room temperature and the product was filtered and washed with water until neutral and air dried. Yield: 0.33 g (0.7 mmol, 88 %). The final product was used without further purification. MS (DUIS): calculated MW 470.13. Found: (-) 469 (M-1), (+) 471 (M+1). 1H NMR (400 MHz, DMSO-da) 5 12.71 (s, 1H), 8.08 (dt, J = 7.6, 1.7 Hz, 1H), 8.03 - 7.92 (m, 1H), 7.64 - 7.52 (m, 2H), 7.45 - 7.39 (m, 1H), 7.39 -7.27 (m, 2H), 7.19 (dd, J = 6.9, 2.3 Hz, 1H), 6.70 (d, J = 1.9 Hz, 1H), 6.69 - 6.61 (m, 2H), 3.47 (q, J = 7.2 Hz, 4H), 1.23 - 1.05 (m, 6H).

Example 7-2

2-(7-(Diethylamino)-2-oxo-3-(6-sulfobenzothiazol-2-yl)-2H-chromen-4-yl)benzoic acid (compound I-13).

2-[3-(Benzothiazol-2-yl)-7-(diethylamino)-2-oxo-2H-chromen-4-yl]benzoic acid (compound I-1) (1.3 g, 2.76 mmol) was placed in a round-bottomed flask, dried in vacuo, then cooled in a dry ice-acetone bath. Fuming sulfuric acid (20%, 5.8 mL, 27 mmol) was added dropwise while stirring. This reaction mixture was stirred at room temperature for 1 hour, then heated for 1 hour at 50 °C and the starting material dissolved. The reaction mixture was cooled and anhydrous ether was added cautiously. The whitish hygroscopic precipitate was filtered off and the filtrate was again triturated with ether and then EtOH. A pink precipitate was collected. Yield 1.21 g (80 %). The final product was used without further purification. MS (DUIS): calculated MW 550.09. Found: (-) 549 (M-1), (+) 551 (M+1). H NMR (400 MHz, TFA) 58.85 - 8.78 (m, 1H), 8.69 (s, 1H), 8.57 - 8.45 (m, 2H), 8.26 - 8.15 (m, 2H), 7.94 (s, 1H), 7.67 (d, J = 9.4 Hz, 1H), 7.63 - 7.56 (m, 1H), 7.45 (dd, J = 8.8, 4.5 Hz, 1H), 3.94 (q, J = 7.2 Hz, 4H), 1.38 (t, J = 7.1 Hz, 6H). According to HPLC and NMR spectra analysis, this product consists mainly of one isomer.

Example 7-3

2-(7-(Diethylamino)-2-oxo-3-(6-chlorosulfonylbenzothiazol-2-yl)-2H-chromen-4-yl)benzoic acid (compound I-14).

Chlorosulfonic acid (5 mL, 75 mmol) was cooled in an ice bath and dye I-1 (1.2 g, 2.55 mmol) was carefully added while stirring in portions over a period of 5 minutes. The combined mixture was stirred at room temperature (~20 °C) for 1.5 hours and then heated at 95-100 °C for 6 hours. After cooling to room temperature, the mixture was very slowly added to approximately 20 mL of ice-water mixture. A red solid separated which was quickly filtered and washed with cold water (2 x 30 mL) and dried in vacuo. Yield 1.2 g (83%, 2.11 mmol). This compound was used in the next step without further purification.

Example 7-4

2-(7-(Diethylamino)-2-oxo-3-(6-aminosulfonylbenzothiazol-2-yl)-2H-chromen-4-yl)benzoic acid (compound I-15).

2-(7-(Diethylamino)-2-oxo-3-(6-chlorosulfonylbenzothiazol-2-yl)-2H-chromen-4-yl) benzoic acid (I-14) (1.2 g, 2.11 mmol) was carefully added portionwise with stirring to an ammonia solution (5 mL, 25%) cooled in an ice bath over a period of 5 min. The reaction mixture was stirred at room temperature (~20 °C) for 1 h and the solvent was distilled off at room temperature in vacuo. The compound was purified by flash column (silica gel, DCM-MeOH as eluent). Yield 0.34 g (30%, 0.62 mmol). This compound contains a small amount of other isomers and was used in the next step without further purification. MS (DUIS): calculated MW 549.10. Found: (-) 548 (M-1), (+) 550 (M+1). 1H NMR (400 MHz, TFA) 5 8.83-8.73 (m, 1H), 8.62 (d, J = 1.6 Hz, 1H), 8.57 (d, J = 8.9 Hz, 1H), 8.42 (dd, J = 8.9, 1.8 Hz, 1H), 8.24-8.13 (m, 2H), 7.82 (d, J = 2.1 Hz, 1H), 7.64-7.52 (m, 2H), 7.38 (d, J = 8.8 Hz, 1H), 3.90 (q, J = 7.2 Hz, 4H), 1.37 (t, J = 7.2 Hz, 6H).

Example 8

4-Nitrophenyl 2-[3-(benzothiazol-2-yl)-7-(diethylamino)-2-oxo-2H-chromen-4-yl]benzoate (compound I-1B)

In anhydrous DMF (1.5 mL) was dissolved 94 mg (0.2 mmol, 1 eq) of 2-[3-(Benzothiazol-2-yl)-7-(diethylamino)-2-oxo-2H-chromen-4-yl]benzoic acid (1-1, NR440). To this solution was added dicyclohexylcarbodiimide (62 mg, 0.3 mmol, 1.5 eq.). This reaction mixture was stirred at room temperature for 3 hours and then p-nitrophenol (33 mg, 0.24 mmol, 1.2 eq.) was added. The reaction mixture was stirred overnight, then dicyclohexylurea was filtered off. The solvent was distilled off in vacuo at room temperature and the oily residue was poured onto ice (ca. 5 g). The yellow solid product was filtered off, washed with water. Yield: 79 mg (67%, 0.134 mmol). MS (DUIS): calculated MW 591.15. Found: (-) 590 (M-1), (+) 592 (M+1).

Example 9

3-(2-(10-(benzothiazol-2-yl)-11 -oxo-2,3,6,7-tetrahydro-1 H,5H,11 H-pyrano[2,3-f]piridor[3, Triethylammonium 2,1-yl]quinolin-9-yl)-N-(4-(tertbutoxy)-4-oxobutyl)benzamido)propane-1-sulfonate (compound1-9A')

Compound I-4 (150 mg, 0.3 mmol, 1 eq) was dissolved in anhydrous DMA (1.5 mL). To this solution was added excess triethylamine (1 mL) and [(1H-benzotriazol-1-yl)oxy]tris(dimethylamino)phosphonium tetrafluoroborate (140 mg, 0.36 mmol, 1.2 eq) as an activating reagent. This reaction mixture was stirred at room temperature for about 20 minutes and then 3-((3-(tert-butoxy)carbonyl)amino)propane-1-sulfonic acid (140 mg, 0.5 mmol, 1.6 eq) was added. The progress of the reaction was monitored by TLC (acetonitrile-water, 10%) and by LCMS.

The reaction mixture was stirred overnight, filtered and excess activating reagent was quenched with a 0.1 M solution of TEAB in water. The solvents were evaporated in vacuo and the residue was purified by HPLC. Yield: 180 mg (0.21 mmol, 69 %). MS (DUIS): calculated MW 757.25. Found: (-) 756 (M-1), (+) 758 (M+1) for the corresponding protonated anion compound I-9A:

Example 10

4-(2-(10-(benzothiazol-2-yl)-11 -oxo-2,3,6,7-tetrahydro-1 H,5H,11 H-pyrano[2,3-f]pyrido[3, Triethylammonium 2,1-yl]quinolin-9-yl)-N-(3-sulfonatopropyl)benzamido)butanoate (compound I-9')

Compound I-9A' from the previous step after evaporation of solvents (180 mg, 0.21 mmol) was dissolved in DCM (150 mL) and trifluoroacetic acid (5 mL) was added. The reaction mixture was left at room temperature overnight. The progress of the deprotection reaction was monitored by TLC (acetonitrile-water, 10% as eluent) and by LCMS. DCM was distilled off. The residue was dissolved in acetonitrile and triethylamine (1 mL) was added. The yellow precipitate was filtered off. MS (DUIS): calculated MW 701.19. Found: (-) 700 (M-1) for the corresponding protonated anion compound I-9:

Example 11

3-(2-(3-(benzothiazol-2-yl)-7-(diethylamino)-2-oxo-2H-chromen-4-yl)-N-(4-(tert-butoxy)-4-oxobutyl) triethylammonium benzamido)propane-1-sulfonate (compound I-6A')

2-(3-(Benzothiazol-2-yl)-7-(diethylamino)-2-oxo-2H-chromen-4-yl)benzoic acid (compound I-1) (94 mg, 0.2 mmol, 1 eq) was dissolved in anhydrous DMA (2 mL). To this solution was added an excess of N-ethyl-N,N-diisopropylamine (1 mL). To this solution was added [(1H-benzotriazol-1-yl)oxy]tris(dimethylamino)phosphonium tetrafluoroborate (80 mg, 0.21 mmol, 1.04 eq) as an activating reagent. The reaction mixture was stirred at room temperature.

The progress of the reaction was monitored by TLC (acetonitrile-water, 10% as eluent). Then, 3-((3-(tert-butoxy)carbonyl)amino)propane-1-sulfonic acid (70 mg, 0.25 mmol, 1.25 eq) was added. The reaction mixture was stirred overnight at room temperature, filtered and the excess activating reagent was quenched with a 0.1 M solution of TEAB in water (3 mL). The solvents were evaporated in vacuo and the residue was purified by HPLC. Yield: 113 mg (0.136 mmol, 68%). MS (DUIS): MW calculated 733.25. Found: (-) 732 (M-1) for the corresponding protonated anion compound I-6A:

Example 12

Triethylammonium 4-(2-(3-(benzothiazol-2-yl)-7-(diethylamino)-2-oxo-2H-chromen-4-yl)-N-(3-sulfonatopropyl)benzamido)butanoate (compound 1- 6')

Compound I-6A' (180 mg, 0.21 mmol) from the previous step after evaporation of solvents was dissolved in DCM (50 mL) and trifluoroacetic acid (2.5 mL) was added. The reaction mixture was allowed to stir at room temperature. The progress of the reaction was monitored by TLC (acetonitrile-water, 10% as eluent). The solvent was distilled off and the residue was dissolved in acetonitrile and triethylamine (1.8 mL) was added. Acetonitrile and excess triethylamine were distilled off. To the residue in the flask was added anhydrous diethyl ether and the product formed as a yellow crystalline precipitate and was filtered. MS (DUIS): Calcd MW 677.19. Found: (-)676 (M-1) for acid I-6:

Example 13

4-(2-(10-(benzothiazol-2-yl)-11 -oxo-2,3,6,7-tetrahydro-1 H,5H,11 H-pyrano[2,3-f]pyrido[3, tert-butyl 2,1-yl]quinolin-9-yl)benzamido)butanoate (I-10A)

Compound I-4 (97 mg, 0.196 mmol, 1 eq) was dissolved in freshly distilled anhydrous DMF (2.5 mL). To this solution was added excess N-ethyl-N,N-diisopropylamine (0.253 mg, 1.961 mmol, 10 eq) and [(1H-benzotriazol-1-yl)oxy]tris(dimethylamino)phosphonium tetrafluoroborate (113 mg, 0.294 mmol, 1.5 eq) as an activating reagent. The reaction mixture was stirred at room temperature for about 20 minutes and then tert-butyl 4-aminobutanoate hydrochloride (58 mg, 0.294 mmol, 1.5 eq) was added. The progress of the reaction was monitored by TLC (acetonitrile-water, 10%) and by LCMS.

The reaction mixture was stirred overnight, filtered and the excess activating reagent was quenched with a 0.1 M solution of TEAB in water. The solvents were evaporated in vacuo and the residue was purified by HPLC. Yield: 105 mg (0.165 mmol, 84 %). MS (DUIS): MW calcd 635.25. Found: (-) 634 (M-1), (+) 636 (M+1).

Example 14

4-(2-(10-(benzothiazol-2-yl)-11 -oxo-2,3,6,7-tetrahydro-1 H,5H,11 H-pyrano[2,3-f]pyrido[3) acid ,2,1 -yl]quinolin-9-yl)benzamido)butanoic acid (compound I-10)

Compound I-10A' (64 mg, 0.1 mmol) from the previous step after evaporation of solvents was dissolved in DCM (25 mL) and trifluoroacetic acid (3.5 mL) was added. The reaction mixture was left at room temperature overnight. The progress of the reaction was monitored by TLC (acetonitrile-water, 10% as eluent) and by LCMS. The solvents were distilled off. The residue was dissolved in acetonitrile and triethylamine (1 mL) was added. Acetonitrile and excess triethylamine were distilled off. The residue in the flask was dried overnight in vacuo, then sonicated with petroleum ester (25 mL) for 1 hour. The yellow precipitate was filtered off. MS (DUIS): calculated MW 579.18. Found: (-) 578 (M-1), (+) 580 (M+1).

Example 15

4-(2-(10-(benzothiazol-2-yl)-11 -oxo-2,3,6,7-tetrahydro-1 H,5H,11 H-pyrano[2,3-f]pyrido[3) acid ,2,1 -yl]quinolin-9-yl)benzamido)butanoic acid (compound I-7)

Compound 1-1B (180 mg, 0.3 mmol, 1 eq) was dissolved in anhydrous DMF (5 mL) and dimethylaminopyridine (56 mg, 0.45 mmol, 1.5 eq) was added. To this reaction mixture was added 4-aminobutanoic acid (47 mg, 0.45 mmol, 1.5 eq) and this reaction mixture was left at room temperature overnight. The progress of the reaction was monitored by TLC (acetonitrile-water, 10% as eluent) and by LCMS. The solvents were distilled off in vacuo at room temperature. The residue was diluted with water (25 mL) and the product formed as a yellow precipitate and was filtered. MS (DUIS): calculated MW 555.18. Found: (-) 554 (M-1), (+) 556 (M+1).

Example 16

2-(3-(5-chlorobenzoxazol-2-yl)-7-(diethylamino)-2-oxo-2H-chromen-4-yl)benzoic acid (compound I-16)

A mixture of 0.313 g (1.0 mmol, 1 eq) of 2-[4-(diethylamino)-2-hydroxybenzoyl]benzoic acid and 0.263 g (1.1 mmol, 1.1 eq) of ethyl 2-(5-chlorobenzoxazol-2-yl)acetate was added with stirring to sulfuric acid (5 mL) at room temperature (ca. 20 °C). This reaction mixture was stirred at room temperature for 3 hours, then heated for 4 hours while stirring at 100 °C. The progress of the reaction was monitored by TLC (DCM-MeOH, 10% as eluent) and by LCMS. The reaction mixture was left at room temperature (ca. 20 °C) overnight and then poured into a mixture of water with crushed ice (ca. 100 g) and sodium acetate (ca. 5 g). After 1 hour, the product was filtered and washed with water until neutral reaction (pH ca. 7) and air dried. Yield: 0.453 g (0.93 mmol, 93 %). The final product was used without further purification. MS (Du IS): MW calculated 488.11. Found: (-) 487 (M-1 ).

Example 17

General procedure for the synthesis of fully functional nucleotide conjugates

Novel dye-nucleotide conjugates were synthesized from compounds (I) or compounds (VO) by coupling with appropriate nucleotide derivatives.

Anhydrous DMA (2.5 mL), W,W,W'W'-tetramethyl-O-(W-succinimidyl)uronium tetrafluoroborate (20 mg, 60 ^mol) and Hunig's base (0.05 mL) were added to the dry mixture of the appropriate dye (50 ^mol). The reaction mixture was stirred at room temperature. According to TLC (acetonitrile-water, 10%), the activation was usually completed in 20-30 minutes. After the activation was complete, the solution of pppC-LN3 as a triethylammonium salt (55 ^mol) in a mixture of DMA (0.75 mL) and water (0.30 mL) was added to the reaction mixture. The reaction mixture was stirred at room temperature under a nitrogen atmosphere overnight. The reaction mixture was cooled to approximately 4 °C with an ice bath, then a solution of 0.1 M TEAB (5 mL) in water was added and the mixture was stirred at room temperature for 10 min. The reaction mixture was applied to a column with approximately 25 g of DEAE Sephadex resin suspension in 0.05 M TEAB solution in water and washed with TEAB (concentration gradient from 0.1 M to 1 M). The colored fractions were collected and evaporated, then coevaporated again with water to remove more TEAB and aspirated to dryness. The residue was then re-dissolved in 0.1 M TEAB. This solution was filtered through a syringe filter with a pore size of 0.2 nm. The product was purified by HPLC using a C18 reverse phase column with acetonitrile-0.1 M TEAB. Yield: 60-75%.

Using the general procedure described above, the following nucleotide conjugates were prepared:

Example 18

In this example, the spectral properties of several fluorescent dyes described herein and the corresponding nucleotide conjugates were tested and compared to commercially available dyes.

Table 1A illustrates the spectral properties of the novel fluorescent dyes described herein in comparison to some exemplary commercial dyes with absorption in the same spectral region in ethanol solution. It can be seen that each of the novel fluorescent dyes of the present application has a larger Stokes shift compared to Star440sx.

Table 1A. Comparison of spectral properties of varying dyes in EtOH solution

In addition, the spectral properties of dye 1-1 were compared with the sulfonated dyes I-13 and I-15 in methanol solution and the results were summarized in Table 1B. These data demonstrate that the sulfonated derivatives I-13 and I-15 have a 2- to 5-fold stronger fluorescence in solution compared to the corresponding unsubstituted analog I-1. These results demonstrate the potential use of sulfonated dyes as biomarkers.

Table 1B. Comparison of the spectral properties of sulfonated dyes in methanol solution

Figure 1 illustrates the fluorescent spectra of the novel fluorescent dyes of the present application in comparison to a commercial dye Star440sx that has absorption in the same spectral region (universal scanning mixture solution; dyes were excited at 460 nm). Due to the higher Stokes shift, these newly developed dyes can provide more signal in the detection region with better separation of the excitation light at 460 nm.

Table 2 illustrates the relative fluorescent intensities of the C-nucleotide labeled with new dyes compared to the appropriate commercial dye-labeled nucleotide having absorption in the same spectral region. It is demonstrated that the new fluorescent dyes described herein can provide 60-400% more signal at 570 nm (detection region) when excited with light at 460 nm due to their stronger fluorescence and larger Stokes shift.

Table 2. Relative fluorescence intensities of the exemplary fully functional C-labeled nucleotide

Table 3 illustrates the spectral properties of C-nucleotides labeled with the novel fluorescent dyes described herein in solution, which is consistent with the observation in Table 1 that most of the nucleotides labeled with the fluorescent dyes of the present application have a larger Stokes shift compared to the commercial dye for the same spectral region.

Table 3. Spectral properties of an exemplary fully functionalized and labeled C-nucleotide in solution.

Figure 2 illustrates the usability of the C-nucleotide labeled with the novel fluorescent dyes described herein (shown in black) for sequencing analysis. In this sequencing example, the dual-channel detection method was used. With respect to the dual-channel methods described herein, nucleic acids can be sequenced using methods and systems described in patent application publication US-2013/0079232. In dual-channel detection, a nucleic acid can be sequenced by providing a first type of nucleotide that is detected in a first channel, a second type of nucleotide that is detected in a second channel, a third type of nucleotide that is detected in both the first and second channels, and a fourth type of nucleotide that lacks a label that is not, or minimally, detected in either channel.

In each of Figures 2A and 2B, the “G” nucleotide is unlabeled and is shown as the lower left cloud. There is a mixture of two “A” labeled nucleotides, one labeled with a coumarin-derived dye at 0.5 pM and one with the benzopyran derivativeI-6a at 1.5 pM shown as the upper right cloud. The signal from the “T” dye-labeled nucleotide NR550S0 is indicated by the upper left cloud, and the “C” labeled nucleotide signal is indicated by the lower right cloud. The X-axis shows the signal intensity from one channel, and the Y-axis shows the signal intensity from the other channel. In Figures 2A and 2B, ffC is labeled withI-6 and I-1, respectively. Experiment setup: Instrument: Run # M15: C-I-6a C5, C-I-1a C9, Sequencing lib: PhiX standard. It is shown that fully functional C-nucleotide conjugates labeled with I-6 and I-1 dyes provided sufficient signal intensities and improved cloud separation.

Figure 3 illustrates the relative fluorescent intensities of C-nucleotides labeled with various fluorescent dyes when excited with blue (460 nm) or green (530 nm) light. This bar graph shows the raw intensity of clusters that have incorporated the indicated dye. Each measurement was taken manually at Ex460 nm and exposing for 1 second at 60 °C. MTS [MiSeq R&D (Test) Software] on M15 was used to take the images, and the Firecrest image analysis tool was used to extract the intensity data.

The results show that ffC labeled with the novel fluorescent dyes described herein provided up to 300% brighter signal compared to the commercial long Stokes dye for the same spectral region.

Figure 4 illustrates the relative brightness of C-conjugated nucleotides labeled with a commercial and a novel fluorescent dye I-6 when excited with blue light (460 nm) at two different temperatures (room temperature at 22 °C and elevated temperature at 60 °C). ffC labeled with the novel fluorescent dye I-6 gave a brighter signal compared to ffC labeled with the commercial long Stokes dye Star440ls for a certain spectral region at both temperatures.

Claims (1)

CLAIMS A nucleotide or oligonucleotide labeled with a fluorescent compound of the formula (V): wherein each R1, R1' and R2 is independently selected from the group consisting of H, alkyl, substituted alkyl, alkoxy, alkenyl, alkynyl, haloalkyl, haloalkoxy, alkoxyalkyl, amino, aminoalkyl, aminosulfonyl, halo, cyano, hydroxy, hydroxyalkyl, heteroalkyl, C-carboxy, O-carboxy, C-amido, N-amido, nitro, sulfonyl, sulfo, sulfino, sulfonato, sulfonyl halide, S-sulfonamido, N-sulfonamido, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heteroaryl and optionally substituted heterocyclyl; Alternatively, R1 and R1' together and with the atoms to which they are attached form a ring or ring system selected from the group consisting of optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocyclyl; each R3 and R4 is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, alkynyl, aminoalkyl, haloalkyl, heteroalkyl, alkoxyalkyl, sulfo, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted carbocyclyl, and optionally substituted heterocyclyl; alternatively, R1 and R3 together with the atoms to which they are attached form a ring or ring system selected from the group consisting of optionally substituted 5-10 membered heteroaryl or optionally substituted 5-10 membered heterocyclyl; Alternatively, R2 and R4 together with the atoms to which they are attached form a ring or ring system selected from the group consisting of optionally substituted 5-10 membered heteroaryl and optionally substituted 5-10 membered heterocyclyl; RHet is a heteroaryl optionally substituted with one or more R5; each R5 and R6 is independently selected from the group consisting of alkyl, substituted alkyl, alkoxy, alkenyl, alkynyl, haloalkyl, haloalkoxy, alkoxyalkyl, amino, aminoalkyl, aminosulfonyl, halo, cyano, hydroxy, hydroxyalkyl, heteroalkyl, C-carboxy, O-carboxy, C-amido, N-amido, nitro, sulfonyl, sulfo, sulfino, sulfonato, sulfonyl halide, S-sulfonamido, N-sulfonamido, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted heterocyclyl; R is selected from -OR7 or -NR8R9; Y is selected from O or NH; R7 is selected from the group consisting of H, alkyl, substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted carbocyclyl, and optionally substituted heterocyclyl; each R8 and R9 is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, alkynyl, aminoalkyl, carboxyalkyl, sulfonatoalkyl, haloalkyl, heteroalkyl, alkoxyalkyl, sulfo, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted carbocyclyl, and optionally substituted heterocyclyl; and n is an integer from 0 to 4, where substituted means that the group is substituted with one or more substituents independently selected from C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 heteroalkyl, C3-C7 carbocyclyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), C3-C7 carbocyclyl-C1-C6 alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 3-10 membered heterocyclyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, ... C1-C6 and C1-C6 haloalkoxy), 3-10 membered heterocyclyl-C1-C6 alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), aryl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), aryl-(C1-C6)alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 5-10 membered heteroaryl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 5-10 membered heteroaryl (C1-C6)alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), halo, cyano, hydroxy, C1-C6 alkoxy, C1-C6 alkoxy-(C1-C6)alkyl (i.e. ether), aryloxy, sulfhydryl (mercapto), halo-(C1-C6)alkyl (e.g. -CF3), halo-alkoxy (C1-C6) (e.g. -OCF3), C1-C6 alkylthio, arylthio, amino, amino(C1-C6)alkyl, nitro, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, acyl, cyanate, isocyanate, thiocyanate, isothiocyanate, sulfinyl, sulfonyl, sulfo, sulfino, sulfonate, and oxo (=O); and wherein the alkyl group has 1 to 20 carbon atoms, the alkenyl group has 2 to 20 carbon atoms, the alkynyl group has 2 to 20 carbon atoms, the heteroalkyl group has 1 to 20 carbon atoms, and the aryl group has 6 to 18 carbon atoms 2. The nucleotide or oligonucleotide of claim 1, wherein RHet is selected from optionally substituted 5- to 10-membered heteroaryl. 3. The nucleotide or oligonucleotide of claim 1 or 2, wherein RHet is selected from optionally substituted 9-membered heteroaryl. 4. The nucleotide or oligonucleotide of any one of claims 1 to 3, wherein RHet is optionally substituted 2-benzothiazolyl with the structure or 2-benzoxazolyl optionally substituted with the structure 5. The nucleotide or oligonucleotide of any one of claims 1 to 4, wherein Y is O. 6. The labeled nucleotide or oligonucleotide of any one of claims 1 to 5, wherein the compound is covalently linked to the nucleotide or oligonucleotide through -C(=O)R, wherein R is -OR 7 , and R 7 is a substituted alkyl. 7. The labeled nucleotide or oligonucleotide of any one of claims 1 to 5, wherein the compound is covalently linked to the nucleotide or oligonucleotide through -C(=O)R, wherein R is -NR8R9 , and wherein at least one of R 8 or R 9 is a substituted alkyl. 8. The labeled nucleotide or oligonucleotide of any one of claims 1 to 7, wherein the compound is linked to the C5 position of a pyrimidine base or the C7 position of a 7-deazapurine base of the nucleotide or oligonucleotide through a linker moiety. 9. The labeled nucleotide or oligonucleotide of any one of claims 1 to 8, further comprising a 3'-OH blocking group covalently linked to the ribose or deoxyribose sugar of the nucleotide or oligonucleotide. 10. A kit comprising one or more nucleotides, wherein at least one nucleotide is a labeled nucleotide according to claims 1 to 9. 11. The kit of claim 10 comprising two or more labeled nucleotides. 12. The kit of any one of claims 10 or 11, further comprising a third and a fourth nucleotide, wherein each of the second, third and fourth nucleotides is labeled with a different compound, wherein each compound has a distinct absorbance maximum and each of the compounds is distinguishable from the other compounds. 13. A sequencing method comprising incorporating a labeled nucleotide according to any one of claims 1 to 9 into a sequencing assay and detecting the nucleotide. 14. The method of claim 13, wherein the sequencing assay is performed on an automated sequencing instrument, and wherein the automated sequencing instrument comprises two light sources operating at different wavelengths.